Archaeobacterial Ether Lipid Liposomes (Archaeosomes) as Novel Vaccine and Drug Delivery Systems

ABSTRACT:?

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeo-bacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.     

A structural comparison of the total polar lipids from the human archaea Methanobrevibacter smithii and Methanosphaera stadtmanae and its relevance to the adjuvant activities of their liposomes.

Mice were immunized with bovine serum albumin (BSA) entrapped within archaeosomes (i.e. liposomes) composed of the total polar lipids (TPL) from the two methanogenic archaea common to the human digestive tract. Methanobrevibacter smithii archaeosomes boosted serum anti-BSA antibody to titers comparable to those achieved with Freund's adjuvant, whereas Methanosphaera stadtmanae archaeosomes were relatively poor adjuvants. An explanation for this difference was sought by analysis of the polar lipid composition of each archaeobacterium. Fast atom bombardment mass spectrometry and NMR analyses of the purified lipids revealed a remarkable similarity in the ether lipid structures present in each TPL extract. However, the relative amounts of each lipid species varied dramatically. The phospholipid fraction in M. stadtmanae TPL was dominated by archaetidylinositol (50 mol% of TPL) and the glycolipid fraction by beta-Glcp-(1,6)-beta-Glcp-(1,1)-archaeol (36 mol%), whereas in M. smithii extracts, both caldarchaeol and archaeol lipids containing a phosphoserine head group were relatively abundant. Liposomes prepared from purified archaetidylinositol and from M. stadtmanae TPL supplemented with increasing amounts of phosphatidylserine elicited poor humoral responses to encapsulated BSA. A dramatic loss in the adjuvanticity of M. smithii archaeosomes was seen upon incorporation of 36 mol% of the uncharged lipid diglucosyl archaeol and, to a lesser extent, of 50 mol% of archaetidylinositol. Interestingly, the relative rates of uptake of M. smithii and M. stadtmanae archaeosomes by phagocytic cultures in vitro were similar. Thus, the lipid composition may influence archaeosome adjuvanticity, particularly a high diglucosyl archaeol and/or archaetidyl inositol content, resulting in a low adjuvant activity.     

Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice

Intranasal administration of ovalbumin (OVA) formulated in an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system prepared by the addition of CaCl2 to small unilamellar archaeosomes (liposomes made from archaeal polar lipids) containing encapsulated OVA, was recently shown to elicit strong and sustained OVA-specific mucosal and systemic immune responses. In this study, we show that the centrifugation/washing and antigen quantization steps required in the standard protocol for obtaining OVA/AMVAD model vaccine formulations can be eliminated by using simpler protocols such as admixing OVA with preformed empty archaeosomes, or by changing the starting ratio (w/w) of archaeal lipid to antigen at the archaeosome preparation stage, prior to the addition of CaCl2 to convert to the AMVAD structures. Irrespective of the vaccine preparation protocol, the AMVAD particle typically comprised of larger spherical structures that had aggregated like a bunch of grapes, and it contained aqueous compartment(s). The anti-OVA IgA antibody responses in vaginal wash, nasal wash, serum, and bile samples, and the anti-OVA IgG antibody responses in sera, in mice intranasally immunized with the OVA/AMVAD formulations prepared by the simplified or the standard protocols, were comparable.     

Safety of archaeosome adjuvants evaluated in a mouse model

Archaeosomes, liposomes prepared from the polar ether lipids extracted from Archaea, demonstrate great potential as immunomodulating carriers of soluble antigens, promoting humoral and cell mediated immunity in the vaccinated host. The safety of unilamellar archaeosomes prepared from the total polar lipids (TPL) of Halobacterium salinarum, Methanobrevibacter smithii or Thermoplasma acidophilum was evaluated in female BALB/c mice using ovalbumin (OVA) as the model antigen. Groups of 6-8 mice were injected (0.1 mL final volume) subcutaneously at 0 and 21 days, with phosphate buffered saline (PBS), 11 microg OVA in PBS, 1.25 mg of antigen-free archaeosomes in PBS (ca 70 mg/kg body wt), or PBS containing 11-20 microg OVA encapsulated in 1.25mg archaeosomes. Animals were monitored daily for injection site reactions, body weight,temperature and clinical signs of adverse reactions. Sera were collected on days 1, 2, 22, and 39 for analyses of creatine phosphokinase. Mice were sacrificed on 39 d, sera were collected for biochemical analyses, and major organs (liver, spleen, kidneys, heart, lungs) were weighed and examined macroscopically. There were no indications of adverse reactions or toxicity associated with any of the archaeosome adjuvants. None of the antigen-free archaeosomes elicited significant anti lipid antibodies when subcutaneously injected (1 mg each at 0, 1, 2, and 4 weeks) in mice, although anti H. salinarum lipid antibodies were detected. These antilipid antibodies cross-reacted with the TPL of T. acidophilum archaeosomes but not with the TPL of M. smithii archaeosomes nor with lipids of ester liposomes made from L-alpha-dimyristoylphosphatidylcholine (DMPC), L-alpha-dimyristoylphosphatidylglycerol (DMPG), and cholesterol (CHOL). In vitro hemolysis assay on mouse erythrocytes indicated no lysis with M. smithii or T. acidophilum archaeosomes at up to 2.5 mg/mL concentration. At this concentration, H. salinarum archaeosomes and DMPC/DMPG/CHOL ester liposomes caused about 2% and 4% hemolysis, respectively. Based on this mouse model evaluation, archaeosomes are well-tolerated and appear relatively safe for potential vaccine applications.     

DIMENSIONAL ANALYSIS AND SCALE-UP IN THEORY AND INDUSTRIAL APPLICATION*

A comparative study between archaeosomes, lipid lamellar vesicles made from archaea polar lipids, and conventional phospholipids liposomes was carried out, aiming at evaluating the properties and the potential of archaeosomes as novel colloidal carriers for effective drug delivery to the skin. Betamethasone dipropionate (BMD)–loaded archaeosomes and conventional liposomes were prepared by the thin-lipid film and sonication procedures, using, respectively, archaeal lipids extracted from archaea Halobacterium salinarum and enriched soy phosphatidylcholine. Vesicular formulations were characterized by assessing vesicle size, zeta potential, incorporation efficiency, and morphology. In order to investigate the effect of the incorporation in the two different colloidal carrier systems on the (trans)dermal delivery of BMD, in vitro drug permeation studies through full-thickness pig skin were carried out by using Franz diffusion vertical cells by testing both archaeal and liposomal dispersions. Interestingly, archaeosomes appeared to be the most effective carriers for the model drug, achieveing a major drug penetration and accumulation in the skin strata, especially in the epidermis. This can, presumably, be due to the enhanced archaeosomal bilayer fluidity, as indicated by the rheological studies that provided insight into the viscoelastic properties of all the studied systems. The available data suggest that suitably developed archaeosomes may hold great promise as delivery vehicles for topical applications.     

Uptake of Archaeobacterial Liposomes and Conventional Liposomes by Phagocytic Cells

Abstract

Liposomes in the 200 nm size range were prepared from the ether lipids extracted from various Archaeobacteria (coined archaeosomes), and from conventional lipids. The entrapment of peroxidase or carboxyfluorescein was used to compare the in vitro uptake of various liposomes by murine peritoneal macrophages, J774A.1 macrophages and several non phagocytic cell lines. While liposomes composed of ester lipids dipalmitoylphosphatidylcholine, or dimyristoylphosphatidylcholine: dimyristoylphosphatidylglycerol: cholesterol (1.8:0.2:1.5, molar ratio) were taken up by macrophage species, the uptake of archaeosomes was 3 to 53 times greater. Uptake by non phagocytic HEp-2, HeLa, and EJ/28 cells was considerably less. Evidence from time-course studies using cytochalasins B+D, sub-optimal temperature or formaldehyde treatments of macrophages, indicated that the archaeosomes lost structural integrity following internalization within the J774A. 1 phagocytic cells. No cytotoxicity was observed in viability or growth assays with J774A. 1 cells, using high doses of three representative types of archaeosomes and one type of conventional-liposome. Therefore, archaeosomes may be well suited to applications where phagocytic cells are a target site.     

Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen

Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis of archaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.     

Isopranoid- and dipalmitoyl-aminophospholipid adjuvants impact differently on longevity of CTL immune responses

The success of lipid membranes as cytotoxic T-cell (CTL) adjuvants requires targeted uptake by antigen-presenting cells (APCs) and delivery of the antigen cargo to the cytosol for processing. To target the phosphatidylserine (PS) receptor of APCs, we prepared antigen-loaded liposomes containing dipalmitoylphosphatidylserine and archaeal lipid liposomes (archaeosomes), containing an equivalent amount of archaetidylserine, and compared their ability to promote short and long-term CTL activity in animals. CTL responses were enhanced by the incorporation of PS into phosphatidylcholine/cholesterol liposomes and, to a lesser extent, into phosphatidylglycerol/cholesterol liposomes, that correlated to the amount of surface amino groups reactive with trinitrobenzoyl sulfonate. Archaeosomes contrasted to the liposome adjuvants by exhibiting higher amounts of surface amino groups and inducing superior shorter and, especially, longer-term CTL responses. The incorporation of dipalmitoyl lipids into archaeosomes induced instability and prevented long-term, but not short-term, CTL responses in mice. The importance of glycero-lipid cores (isopranoid versus dipalmitoyl) to the longevity of the CTL response achieved was shown further by incorporating dipalmitoyl phosphatidylethanolamine (DPPE) or equivalent amounts of synthetic archaetidylethanolamine (AE) into archaeosome adjuvants. Both DPPE and AE at equivalent (5 mol%) concentrations enhanced the rapidity of CTL responses in mice, indicating the importance of the head group in the short term. In the longer term, 5% of DPPE (but not 5% of AE) was detrimental. In addition to head-group effects critical to the potency of short-term CTL responses, the longer term CTL adjuvant properties of archaeosomes may be ascribed to stability imparted by the archaeal isopranoid core lipids.     

Bipolar tetraether archaeosomes exhibit unusual stability against autoclaving as studied by dynamic light scattering and electron microscopy

The stability of liposomes made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius against autoclaving has been studied by using dynamic light scattering and transmission electron microscopy. PLFE lipids have structures distinctly different from those derived from eukaryotes and prokaryotes. PLFE lipids are bipolar tetraether molecules and may contain up to four cyclopentane rings in each of the two dibiphytanyl chains. In the pH range 4-10, PLFE-based archaeosomes, with and without polyethyleneglycol- and maleimide-lipids, are able to retain vesicle size, size distribution, and morphology through at least six autoclaving cycles. The cell growth temperature (65 °C vs. 78 °C), hence the number of cyclopentane rings in the hydrocarbon chains, does not affect this general conclusion. By contrast, at the same pH range, most conventional liposomes made of monopolar diester lipids and cholesterol or pegylated lipids cannot withhold vesicle size and size distribution against just one cycle of autoclaving. At pH < 4, the particle size and polydispersity of PLFE-based archaeosomes increase with autoclaving cycles, suggesting that aggregation or membrane disruption may have occurred at extreme acidic conditions during heat sterilization. Under high salt conditions, dye leakage from PLFE archaeosomes due to autoclaving is significantly less than that from pegylated liposomes composed of conventional lipids. The ability to maintain vesicle integrity after multiple autoclaving cycles indicates the potential usefulness of utilizing PLFE-based archaeosomes as autoclavable and durable drug (including genes, peptides, vaccines, siRNA) delivery vehicles.     

Liposomes in immunology

Liposomes, the artificial phospholipid vesicles, have the capacity of entrapping water soluble substances in their aqueous compartments. Of the many possible potentials of liposomes their application in immunology is most significant. Recent studies have shown an adjuvant and a carrier effect of liposomes to a number of antigens. Liposomes used in these studies are generally multilamellar vesicles with the antigen encapsulated in the aqueous phase. Some antigens may also be associated with the lipid lamellae covalently or noncovalently. The adjuvant property of liposomes is greatly affected by the surface charge of the vesicle as well as the site of association of the antigen. The other factors which may have a role in immunopotentiation by liposomes are the size and structure of the vesicles, the lipid composition, route of administration and their surface sugars. In addition, liposomes may function as carriers to haptens and other antigens. In association with liposomes the nature of the immune response may be modulated. For a further enhancement of the adjuvant activity of liposomes use has been made of immunomodulators.     

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.     

Fluidity enhancement: a critical factor for performance of liposomal transdermal drug delivery system

Liposomes are well known lipid carriers for drug delivery of bioactive molecules encapsulated inside their membrane. Liposomes as skin drug delivery systems were initially promoted primarily for localized effects with minimal systemic delivery. Subsequently, a novel vesicular system, transferosomes was reported for transdermal delivery with efficiency similar to subcutaneous injection. The multiple bilayered organizations of lipids applied in these vesicles structure are somewhat similar to complex nature of stratum corneal intercellular lipids domains. The incorporation of novel agents into these lipid vesicles results in the loss of entrapped markers but it is similar to fluidization of stratum corneum lipids on treatment with a penetration enhancer. This approach generated the utility of penetration enhancers/fluidizing agents in lipids vesicular systems for skin delivery. For the transdermal and topical applications of liposomes, fluidity of bilayer lipid membrane is rate limiting which governs the permeation. This article critically reviews the relevance of using different types of vesicles as a model for skin in permeation enhancement studies. This study has also been designed to encompass all enhancement measurements and analytical tools for characterization of permeability in liposomal vesicular system.     

In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery

Abstract

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer.     

Evaluation of tetraether lipid-based liposomal carriers for encapsulation and retention of nucleoside-based drugs

Although liposomal nanoparticles are one of the most versatile class of drug delivery systems, stable liposomal formulation of small neutral drug molecules still constitutes a challenge due to the low drug retention of current lipid membrane technologies. In this study, we evaluate the encapsulation and retention of seven nucleoside analog-based drugs in liposomes made of archaea-inspired tetraether lipids, which are known to enhance packing and membrane robustness compared to conventional bilayer-forming lipids. Liposomes comprised of the pure tetraether lipid generally showed improved retention of drugs (up to 4-fold) compared with liposomes made from a commercially available diacyl lipid. Interestingly, we did not find a significant correlation between the liposomal leakage rates of the molecules with typical parameters used to assess lipophilicity of drugs (such logD or topological polar surface area), suggesting that specific structural elements of the drug molecules can have a dominant effect on leakage from liposomes over general lipophilic character.     

Lipid Dependent Cardio- Haemodynamic Tolerability of Liposomes in Rats

Abstract

Rationale and Objectives:

The use of contrast-carrying liposomes in diagnostic applications (1) or of haemoglobin liposomes in blood replacement therapy (2) requires infusion of large lipid doses. Saturated lipids like HSPC are often used in these formulations to render the liposomes more stable (3). Previous studies have indicated that intravenous injection of such liposome preparations can result in significant haemodynamic changes in rats (14). The purpose of this study was to systematically evaluate cardio- and haemodynamic effects of liposomes prepared from saturated and unsaturated phosphatidylcholine alone or in combination with other lipid components.

Methods;

Liposomes made from SPC, HSPC, DSPC, DSPC/CH, DSPC/DSPG, DSPC/CH/DSPG were infused in anaesthetized rats (total lipid dose: 300 mg lipid/kg BW) and cardio-heamodynamic parameters were measured.

Results:

DSPC-liposomes significantly reduced blood pressure (BP) and total peripheral resistance (TPR) by -53.7 % and -45.7 % of prevalue, respectively. Similar results were obtained for HSPC-liposomes. Marked ECG-changes were recorded for both formulations. SPC-liposomes caused a transient and moderate reduction of BP and TPR (-17.0 % and -22.3 %, respectively). Short-lasting ECG changes were also observed. The addition of cholesterol or DSPG to DSPC liposomes reduced cardiac and haemodynamic side effects in rats.

Conclusion;

The lipid composition of liposomes is of major importance for the incidence of cardiovascular side effects in rats. Liposomes composed of pure saturated phosphatidylcholine cause significant changes which can be diminished by the addition of other lipid components like cholesterol.     

Stability of Liposomes Prepared from the Total Polar Lipids of Methanosarcina Mazei is Affected by the Specific Salt form of the Lipids

Abstract

The total polar lipids (TPL) extracted from the archaeobacterium Metha-nosarcina mazei were predominantly in the form of sodium and potassium salts. Upon passage of this natural salt form of TPL (ns-TPL) through a silica gel G column (s-TPL), the molar ratio of the monovalent cations, sodium plus potassium, to that of the divalent cations, magnesium plus calcium, decreased from about 11:1 to 2:1. Liposomes (archaeosomes) made from ns-TPL were unable to efficiently retain low molecular weight aqueous markers such as 5(6)-carboxyfluorescein (CF). In phosphate buffered saline (PBS, pH 7.14), between 60-90% and 90-100% of entrapped CF leaked out during 21 h storage at room temperature (20-22°) and 3 h at 50°, respectively. However, archaeosomes made with the s-TPL, as well as from TPL converted to the salt forms of predominantly sodium, potassium, calcium or magnesium, were significantly less leaky. The archaeosomes made with s-TPL were the most stable, showing less than 7% leakage after 21 h at room temperature and only 14% leakage of CF after 3 h at 50°. The stability of s-TPL archaeosomes (21 h, 20-22°) was not affected when Tris-HCI buffer (pH 7.4) was used instead of PBS. However, the inclusion of 1 or 10 mM EGTA in the Tris-HCI buffer increased the amount of CF leaked, to about 25% and 100%, respectively. Except for some differences in phosphatidylserine and phosphati-dylglycerol, the lipid compositions of ns-TPL, s-TPL, and the magnesium form of TPL were similar, as determined by thin layer chromatography of labeled lipids. Archaeosomes prepared from s-TPL and ns-TPL had -“P NMR spectra that were similar to each other, but distinct from those of vesicles prepared from the ester lipid dimyristoylphosphatidylcholine. The types and relative proportions of cations associated with the lipids of M. mazei prior to their hydration and vesicle formation have a major influence, although other factors such as lipid composition may have an effect, on the permeability of the bilayer to low molecular weight compounds.     

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles

A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid from the outer leaflet. Asymmetrically labeled vesicles were separated from albumin/lipid complexes by gel filtration chromatography. Vesicles prepared in this manner were unable to transfer fluorescent lipid to cells during liposome-cell incubations. Liposomes asymmetrically labeled with other 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-phospholipid analogues were also prepared. Removal of amino-dodecanoyl-NBD-labeled lipids from the outer leaflet of liposomes required three times more bovine serum albumin, and 48 h of incubation. This method can be used to produce large amounts of asymmetrically labeled liposomes suitable for use in investigating a variety of membrane phenomena.     

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

Biophysical aspects of using liposomes as delivery vehicles

Liposomes are used as biocompatible carriers of drugs, peptides, proteins, plasmic DNA, antisense oligonucleotides or ribozymes, for pharmaceutical, cosmetic, and biochemical purposes. The enormous versatility in particle size and in the physical parameters of the lipids affords an attractive potential for constructing tailor-made vehicles for a wide range of applications. Some of the recent literature will be reviewed here and presented from a biophysical point of view, thus providing a background for the more specialized articles in this special issue on liposome technology. Different properties (size, colloidal behavior, phase transitions, and polymorphism) of diverse lipid formulations (liposomes, lipoplexes, cubic phases, emulsions, and solid lipid nanoparticles) for distinct applications (parenteral, transdermal, pulmonary, and oral administration) will be rationalized in terms of common structural, thermodynamic and kinetic parameters of the lipids. This general biophysical basis helps to understand pharmaceutically relevant aspects such as liposome stability during storage and towards serum, the biodistribution and specific targeting of cargo, and how to trigger drug release and membrane fusion. Methods for the preparation and characterization of liposomal formulations in vitro will be outlined, too.