Cholesterol inclusion in liposomes affects induction of antigen-specific IgG and IgE antibody production in mice by a surface-linked liposomal antigen

In the previous study, we investigated the induction of ovalbumin (OVA)-specific antibody production in mice by OVA-liposome conjugates made using four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids. All of the OVA-liposome conjugates tested induced IgE-selective unresponsiveness. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes with the highest membrane fluidity, suggesting that the membrane fluidity of liposomes affects their adjuvant effect. In this study, liposomes with five different cholesterol inclusions, ranging from 0% to 43% of the total lipid, were made, and the induction of OVA-specific antibody production by OVA-liposome conjugates was compared among these liposome preparations. In contrast to the results in the previous study, liposomes that contained no cholesterol and possessed the lowest membrane fluidity demonstrated the highest adjuvant effect for the induction of IgG antibody production. In addition, when the liposomes with four different lipid compositions were used, OVA-liposome conjugates made using liposomes that did not contain cholesterol induced significantly higher levels of anti-OVA IgG antibody production than did those made using liposomes that contained cholesterol and, further, induced significant production of anti-OVA IgE. These results suggest that cholesterol inclusion in liposomes affects both adjuvanticity for IgG production and regulatory effects on IgE synthesis by the surface-coupled antigen of liposomes.     

Surface-linked liposomal antigen induces ige-selective unresponsiveness regardless of the lipid components of liposomes

We have previously reported that antigen coupled with liposomes induced antigen-specific and IgE-selective unresponsiveness in mice. This antigen preparation was investigated for application in a novel vaccine protocol to induce minimal IgE synthesis. In this study, ovalbumin (OVA)-liposome conjugates were made using liposomes of four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids, after which the induction of anti-OVA antibody production was investigated in mice. All of the OVA-liposome conjugates induced IgE-selective unresponsiveness. The membrane fluidity of liposomes, as measured by detecting changes in the fluorescence polarization of a 1,6-diphenyl-1,3,5-hexatriene (DPH) probe located in the bilayers, was significantly higher in liposomes consisting of unsaturated carrier lipids than those of the other liposomes consisting of saturated carrier lipids. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes consisting of unsaturated carrier lipids. In addition, among these OVA-liposomes, the one possessing the longest carbon chain induced the lowest IgG antibody production. These results suggest that the membrane fluidity of liposomes might affect the adjuvant effect of liposomes but not the induction of IgE-selective unresponsiveness in immunizations with surface-linked liposomal antigens.     

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

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

Essential oils encapsulated in liposomes: a review

Abstract

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

The Effect of Cholesterol in the Liposome Bilayer on the Stabilization of Incorporated Retinol

The effect of cholesterol in the liposome bilayer on the stability of incorporated retinol was studied. Retinol was incorporated into liposomes containing soybean phosphatidylcholine (PC) and cholesterol (CH) at various ratios, and the liposomes were prepared as multilamellar vesicles by the dehydration–rehydration method. Retinol readily incorporated into liposomes at a ratio of 0.01:1 (w/w) retinol:lipid, with over 94.52% being incorporated in all conditions studied. The incorporation efficiency of retinol increased slightly with increasing CH content in the liposome and with increasing pH of the hydration buffer. Average particle size increased as the CH content increased, and mean particle sizes at pH 5, 7, and 9 were 30.27, 89.53, and 41.42 µm, respectively. The time course of retinol degradation in aqueous solution in liposomes with various ratios of PC to CH was determined under a variety of pH conditions (pH 5, 7, and 9), and temperatures (4, 25, 37, and 50°C). The stability of incorporated retinol was enhanced by increasing the CH content. At pH 7.0 and 4°C, for example, 90.17% of the retinol in liposomes containing 50:50 (PC:CH) remained after 10 days of storage, whereas 51.46% remained at 100:0 (PC:CH). These results indicate that CH in liposomes greatly increases the incorporation efficiency of retinol and the stability of incorporated retinol.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Influence of phospholipid composition on the adjuvanticity and protective efficacy of liposome-encapsulated Leishmania donovani antigens

In this study, we evaluate the effect of phospholipid on the adjuvanicity and protective efficacy of liposome vaccine carriers against visceral leishmaniasis (VL) in a hamster model. Liposomes prepared with distearyol derivative of L-alpha-phosphatidyl choline (DSPC) having liquid crystalline transition temperature (Tc) 54 C were as efficient as dipalmitoyl (DPPC) (Tc 41 C) and dimyristoyl (DMPC) (Tc 23 C) derivatives in their ability to entrap Leishmania donovani membrane antigens (LAg) and to potentiate strong antigen-specific antibody responses. However, whereas LAg in DPPC and DMPC liposomes stimulated inconsistent delayed type hypersensitivity (DTH) responses, strong DTH was observed with LAg in DSPC liposomes. The heightened adjuvant activity of DSPC liposomes corresponded with 95% protection, with almost no protectivity with LAg in DPPC and DMPC liposomes, 4 mo after challenge with L. donovani. These data demonstrate the superiority of DSPC liposomes for formulation of L. donovani vaccine. In addition, they demonstrate a correlation of humoral and cell-mediated immunity with protection against VL in hamsters.     

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: Effect of charge,fluidity and antigen-to-lipid ratio

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6′-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic α-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that α-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

Effect of dehydration and rehydration of the pH-sensitive liposomes containing chimeric gag-V3 virus like particle on their long-term stability

One of the practical limitations with the use of liposomes for delivery of the pharmaceutical substances such as antigens is that liposomes are relatively unstable in storage. In order to extend the stability of liposome in storage without affecting their functional activity, solution-type liposomes were dehydrated to form a structurally intact dry liposomes. Comparative immunological evaluation was carried out for both dry and solution-type liposomes containing gag-V3 chimera, consequently it was found that dry liposomes elicited both humoral and cellular response as efficiently as solution-type liposomes did against the same gag-V3 antigen. Especially, long-term stability of the liposomes was remarkably enhanced by the dehydration made to liposomes without a significant change in its ability to elicit immune responsein vivo. These results indicate that dry pH-sensitive liposome may become an effective delivery and adjuvant system for general vaccine development.     

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.     

Membrane fluidity and lipid hapten structure of liposomes affect calcium signals in antigen-specific B cells.

Antigen-specific B-cell clones directed against a 2,4,6-trinitrophenyl (TNP) hapten have been established [Hamano et al. (1990) J. Immunol. 144, 811-815]. We measured here the cytosolic free calcium ion concentration ([CA2+]i) in these B-cell clones after antigen stimulation. Trinitrophenylated liposomes with different length spacers between TNP and phosphatidylethanolamine (TNP-Cn-PE) increased cytosolic free calcium concentration in TNP-specific B cells (clone TP67.21). The magnitude of calcium signals depended on the length of the spacer. TNP-C6-PE in dipalmitoylphosphatidylcholine (DPPC) liposomes triggered larger calcium signals in B cells than TNP-Cn-PE with n = 0, 4, 8, or 12. The magnitude of the calcium signals was strongly dependent on the fluidity of the liposome membranes. TNP-C6-PE in the solid DPPC liposomes triggered the calcium signals in B cells 50-100 times as efficiently as TNP-C6-PE in the fluid dimyristoylphosphatidylcholine liposomes. The difference between the solid liposomes and the fluid liposomes was more pronounced in triggering calcium signals in B cells than in antibody binding to these liposomes.     

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes

Abstract

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol^-1. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.     

Macrophage Activation by Liposomal Lipid A: Implications for Vaccines

Abstract

Introduction

Vaccine technology has fostered many promising immunologic strategies for disease prevention, especially vaccination with synthetic peptides and recombinant proteins. Such products, even if considered good candidates for vaccines, are often incomplete in that they may require the 'help' of an adjuvant to become immunogenic (1). However, an immunologist considering peptide epitopes for immunization, must in tandem carefully reflect on how the adjuvant directs the immune response. In our experience, while the expected response to an antigen has often been extensively characterized, less attention is given to adjuvant-immune system interactions beyond the desire to enhance the immune response. This is in part due to the lag in studies focusing on adjuvant-immune system interactions when compared to the immunizing antigens themselves, although the study of adjuvants has begun to receive increasing attention. The adjuvant effect on the immune response can be the determining factor in terms of the effector arm of the immune system recruited (e.g., cytoxoxic T lymphocytes, particular IgG subclass antibodies, etc.) (2-4).

Our laboratory has had a long-standing interest in the immune response to liposomal antigens, beginning with phospholipid antibodies (5). This interest has grown and our laboratories are currently participating in several vaccine trials in humans (6-9). This is in large part due to the effectiveness of liposomes containing lipid A in inducing strong antibody and cytotoxic lymphocyte responses to antigens, coupled with minimal or no adverse reactions (6, 10). Our understanding of the mechanisms by which liposomes containing lipid A exert such adjuvant effects is incomplete but is rapidly growing. This review endeavors to highlight some of the known interactions between liposomes containing lipid A and the macrophage that might be of interest to the vaccinologisl, with a particular focus on macrophage activation and the mechanisms by which liposomes containing lipid A cause this to occur.     

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.     

Anti-tumor vaccine adjuvant effects of IL-2 liposomes in mice immunized against MCA-102 sarcoma.

MCA-102, a murine sarcoma previously reported to be non-immunogenic in C57/BL6 murine tumor models was used in a tumor vaccine preparation which included liposome encapsulated IL-2 as an adjuvant. C57/BL6 mice were immunized in the right hind footpad with irradiated MCA-102 murine sarcoma cells on days 0, 7, and 21 with or without IL-2 liposome adjuvant at 25,000 IL-2 units/injection. Mice were challenged with live tumor in the right flank on day 35. Survival of mice given IL-2 liposomes with irradiated MCA-102 cells was significantly prolonged over mice given tumor antigen with saline, and non-immunized mice. In addition, mice which received the IL-2 liposome adjuvant also had prolonged survival over those mice immunized with the additional control adjuvants of free IL-2 or dimyristoyl phosphatidyl choline (DMPC) lipid in the form of empty liposomes. IL-2 liposome plus tumor antigen also yielded a significant local protective response against live MCA-102 tumor challenge. When live tumor was injected into the site of previous immunizations on day 21 after two immunizations, the IL-2 liposome adjuvant group showed significantly delayed local growth of tumor compared to animals immunized without adjuvant, or with the adjuvants of empty liposomes or free IL-2. Finally, immunized mice were challenged with irradiated tumor cells and saline intradermally in the ears and delayed type hypersensitivity (DTH), an indicator of helper T cell response, was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tc's below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tc's above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

Adjuvant effects of nonionic block polymer surfactants on liposome-induced humoral immune response

The ability of several surface-active agents to stimulate the humoral immune response in mice against haptenated liposomes was tested. The surfactants were block copolymers of hydrophilic polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP) that differed in m.w., percentage of POE, and mode of linkage of POP to POE. The liposomes were haptenated with tripeptide-enlarged dinitrophenyl coupled to phosphatidylethanolamine, which was incorporated into the liposomal membrane. Additional injection of mice with surfactant stimulated serum hemagglutination titers and splenic plaque-forming cell (PFC) numbers to varying extents. Block polymers with POP chains flanking a POE center, as well as polymers with POE chains flanking a POP center, displayed high adjuvant activity. These block polymers stimulated the antibody response in a dose-dependent manner. They stimulated the antibody response with both high and low antigen doses. Furthermore, the addition of one of these adjuvants (25R1) reduced the amount of carrier lipid required in the liposome in order to obtain an optimal antibody response. The surfactants, which displayed high adjuvant activity, did not interfere with liposome stability as measured with a liposome lysis assay. Moreover, in vitro preincubation of liposomes with a block polymer did not affect their immunogenicity. Optimal adjuvant activity was observed when both adjuvant and liposomes were administered by the same route. Simultaneous injection of both components, however, is not a prerequisite. Conclusively, it can be stated that nonionic block polymer surfactants are potent adjuvants for stimulation of the antibody response against haptenated liposomes.     

Liposomes in immunology: Evidence that their adjuvant effect results from surface exposition of the antigens

The immune responses against human serum albumin (HSA) and bovine gamma globulin (BGG) were studied in rabbits after intravenous injections of various preparations of these antigens. Antigens were injected free in saline, coated on “empty” liposomes or both coated on liposomes, and entrapped in their inner compartments. The earlier established adjuvant effect of the liposomes was confirmed for both antigens. Although the amount of antigen entrapped in the liposomes was much higher than the amount coated on their outer surfaces, liposomes containing the antigen both in their inner compartments and on their outer surface showed no stronger adjuvant effect than “empty” liposomes coated with the antigen only. The results support the hypothesis that the adjuvant effect of liposomes is mediated by antigens exposed on the outer surfaces of the liposomes. Suggestions are made for the use of liposomes as a practical immunoadjuvant with definite advantages over many other adjuvants.