Incorporation of LPS in liposomes diminishes its ability to induce tumoricidal activity and tumor necrosis factor secretion in murine macrophages

We investigated the effect of lipopolysaccharide (LPS) incorporated into phospholipid vesicles (liposomes) on the induction of macrophage-mediated tumor cytotoxicity and tumor necrosis factor (TNF) secretion. The incorporation of Salmonella minnesota rough (Re)-LPS into multilamellar or small unilamellar vesicles (liposomes) resulted in an 100- to 1,000-fold reduction in its potency to activate both the macrophage cell line RAW 264.7 and murine thioglycolate elicited peritoneal macrophages to become cytotoxic for L929 and P815 tumor cells. Liposomal LPS was also a 100- to 1,000-fold less potent inducer of TNF secretion from RAW 264.7 cells. Cytokines secreted by the activated macrophages contributed to the cytotoxic effect on the L929 cells but not the P815 cell line. Human recombinant TNF was not cytotoxic for either cell line but was cytostatic for the L929 cell line. Morphological examination of the cells after uptake of fluorescent, free, and liposomal LPS revealed that both forms were internalized by the endocytic pathway. This, together with the considerably reduced potency of liposomal LPS to induce tumor cytotoxicity and TNF secretion, suggests that the interaction of the hydrophobic part of the lipid A moiety of LPS with the macrophage plasma membrane is needed to optimally activate these cells. Incorporation of LPS into liposomes effectively abrogates this interaction.     

Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age.     

Altered in vivo activity of liposome-incorporated lipopolysaccharide and lipid A.

We compared the abilities of free and liposome-incorporated Salmonella minnesota wild-type lipopolysaccharide (LPS) and lipid A to activate peritoneal macrophages and induce lethal toxicity in mice. Incorporation of lipid A into multilamellar vesicles resulted in a 100-fold-decreased potency to prime macrophages for phorbol myristate acetate-triggered release of H2O2. In addition, liposome incorporation reduced the lethality of LPS and lipid A at least 10-fold in dactinomycin-sensitized mice. Similar results were obtained with multilamellar liposomes delivered intravenously and when small unilamellar vesicles were employed. The observed difference in toxicity was not dependent on dactinomycin treatment, since a similar decrease was obtained with large doses of liposomal LPS in unsensitized mice. Control liposomes, prepared without LPS and lipid A, did not reduce the activities of the free compounds. The administration of a sublethal amount of liposomal LPS induced within 20 days, but not during the first week, tolerance to a subsequently injected lethal dose of free endotoxin. The latter observation suggests that early-phase tolerance is not the mechanism responsible for the reduced toxicity of liposomal LPS. These data show that liposomal LPS and lipid A have reduced endotoxic activity in vivo and are consistent with our hypothesis that a direct interaction of lipid A with appropriate plasma membrane components is necessary to efficiently trigger biologic responses. This interaction, however, is prevented by the stable insertion of LPS into the liposomal membrane.     

The use of liposomal enrofloxacin for intracellular infections in Kangal dogs and visualization of phagocytosis of liposomes

It is difficult to treat intracellular infections because of the intrinsic resistance of the microorganism to most antibiotics. Moreover, these microorganisms can survive in phagocytic cells (macrophages and neutrophils). In this study, our aims were to encapsulate an antibiotic in liposomes, which will be phagocytized as well as the microorganisms in the phagocytic cell (because liposomes were prepared using lipids which have an antigenic activity and they can be phagocytized, thus, the active substance can be transferred into the cell), and to visualise with microscopy the phagocytic activity of macrophages and neutrophils to liposomes. MLV (multilamellar vesicles) fluorescein-labeled liposomes were prepared and incubated with isolated Kangal shepherd dog macrophages and neutrophils. The phagocytosis of liposomes by monocytes was visualized step by step under the microscope. Liposomes were also observed phagocytized after incubation with neutrophils. Enrofloxacin was chosen as a model drug. Neutrophils and macrophages were isolated from Kangal shepherd dogs and infected with Staphylococcus aureus, and their phagocytic activities (PA) and microbicidal activities (MA) were determined. PA and MA values were redetermined and compared when enrofloxacin formulations were used. Liposomal enrofloxacin was found to be more effective.     

Production of multilamellar, small unilamellar and reverse-phase liposomes containing house dust mite allergens. Potential adjuvants in the immunotherapy of allergic disease

The capacity of multilamellar (MLV), small unilamellar (SUV) and reverse-phase vesicle (REV) liposomes to incorporate house dust mite allergens has been studied. All three liposome preparations entrapped mite proteins with efficiencies of 36% (SUV), 29% (MLV) and 14% (REV). MLV incorporated the complete range of proteins contained in mite extracts with apparent molecular weights ranging from 14,000 to greater than 67,000 as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis. However, several proteins with apparent molecular weights (MW) of 16,000, 36,000 and 43,000 were excluded from the REV and SUV. Immunoblotting analysis using a serum pool prepared from mite allergic individuals showed that whereas the whole spectrum of allergens was incorporated into the MLV, a MW 43,000 allergen was excluded from the REV and SUV. The exclusion of these mite components is probably a function of the relatively prolonged exposure of the original extract to organic solvent in the preparation of the REV and SUV liposomes.     

Enhanced immunogenicity of influenza virus surface proteins when incorporated into liposomes

A method for incorporation of influenza virus surface proteins into monolayer liposomes has been developed on the basis of removal of dialysed non-ionic detergent (beta-octyl glucoside) from a mixture of viral proteins and lipids. The resulting lipid-protein complexes (virosomes) are similar to intact virus particles by their morphological parameters. The immunogenicity of hemagglutinin incorporated into virosomes was significantly higher than that in the preparation of surface proteins isolated from virions which was inoculated into mice with or without complete Freund's adjuvant. The high immunogenicity of hemagglutinin incorporated into liposomes correlated with their high hemagglutinating activity. The possibility of using virosomes as subunit influenza vaccines is discussed.     

Liposomes as vehicle for allergen presentation in the immunotherapy of allergic diseases

Liposomes are non-toxic, biodegradable and weakly immunogenic lipid vesicles which can be used as immunomodulating agents. In the present study, multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) were used to incorporate an allergenic protein from Artemisia scoparia pollen. MLV incorporated more allergenic protein than SUV. To assess the immunomodulating effect of allergen entrapped in liposomes, Swiss strain mice (made IgE responders) were injected with either free allergen or liposome-entrapped allergen (LEA) and their immune response was measured in terms of specific IgG and specific IgE levels. Results indicated that specific IgE response was significantly lower in mice injected LEA (P less than 0.02) than in mice injected free allergenic protein. Although specific IgG response was higher in mice injected LEA, there was no statistically significant difference between the two groups. Potential use of liposomes as non-immunogenic biocompatible vehicle for antigen presentation in immunotherapy will be discussed.     

Heparin incorporating liposomes as a delivery system of heparin from PET-covered metallic stents: effect on haemocompatibility

We investigate the possibility of coating polymer-covered stents with heparin-encapsulating liposomes for improving their haemocompatibility. Thin-film hydration (for multilamellar vesicles, MLV), and the dehydration-rehydration vesicle (DRV) methods are used for preparation of low-molecular weight heparin (LMWH)-encapsulating liposomes with varying lipid compositions. Liposomes are characterized for LMWH encapsulation and retention. For measurement of LMWH, a chromogenic technique is adjusted. For evaluation of heparin release from vesicles in platelet poor plasma (PPP) coagulation time is measured in presence of liposomal samples. Results reveal that LMWH encapsulation in liposomes is higher in DRV, however compositions with high encapsulation are leaky during buffer incubation. Most liposomes release LMWH slowly during plasma incubation (retention after 24 h ranges between 74% and 95%). Concerning the haemocompatibility of polyethylene terephthlate-covered stents after coating with LMWH-encapsulating liposomes, there is a marked increase (higher for DRV-coated stents compared to MLV) in plasma recalcification time compared to the control (plain blood) and reference (non-coated stent), which increases with blood-material contact time. This is probably due to LMWH release, demonstrating that encapsulated LMWH retains its biological functionality. Interestingly, the DRV-coated stents retained a high plasma recalcification time and a large number of liposomes on the stents (as proven by SEM studies) even after extensive washing (high shear conditions), proving that this method may be functional under high flow applying in vivo conditions.     

Lipopolysaccharide (LPS) alters phosphatidylcholine metabolism in elicited peritoneal macrophages

We investigated the effects of LPS on mouse peritoneal macrophage phospholipids using radiolabeled precursors. LPS (200 ng/ml) stimulated incorporation of [32P] into all classes of phospholipids within 0.5 hr, and after 2 hr the increase was 60% greater than controls. Separation of the phospholipid classes by thin-layer chromatography revealed a selective increase in incorporation of label into phosphatidylcholine (PC) (90% increase compared to approximately 50% in the other phospholipids). In macrophages labeled with [3H]-choline, LPS stimulated both the incorporation of label into PC and the release of incorporated label into the medium. The time dependencies of stimulated [3H] release and [32P] incorporation were similar. These data are consistent with the hypothesis that LPS activates macrophages via a PC-specific phospholipase-dependent mechanism.     

Expression ofNeisseria meningitidisclass 1 porin as a fusion protein inEscherichia colli: the influence of liposomes and adjuvants on the production of a bactericidal immune reesponse

High level exxpression of meningococcal class 1 protein was achieved inEscherichia coliusing the p-GEMEX-1 vector, in which the protein was expressed in inclusion bodies, (IB), as a fusion with the bacteriophage T7 gene 10 capsid protein. The fusion protein (FP) was engineered with a factor Xa protease site between the gene 10 and class 1 protein, but treatment with the enzyme resulted in cleavage at additional sites within the class 1 protein. Since it was not possible to remove the leader protein, the intact FP provided an alternative antigen for immunization. Antisera raised to FP, solubilized from IB and incorporated into liposomes, generated a subtype-specific response which was weakly bactericidal for meningococci. In order to remove any possible effect ofE. coliLPS present in IB, the FP was further purified by SDS-PAGE and incorporated into liposomes, either alone ofr in combination with the adjuvants monophosphoryl lipin A or muramyl dipeptide. The incorporation of adjuvants in liposomes resulted in stimulation of the overall immune response to FP, but the resulting antisera were not bactericidal. however and effective bactericidal response was obtained with the purest preparation of FP in liposomes, without any additional adjuvants, revealing that attempts to increase further the immunogenicity of such antigens must not be at the expense of interfering with optimal protein folding     

B Lymphocytes Respond Specifically to Phytohaemagglutinin after Liposome-Dependent Transfer of Purified Phytohaemagglutinin Receptors

Phytohaemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography from porcine splenic lymphocytes, were reconstituted into vesicles made of phosphatidylcholine and phosphatidylserine, concomitantly with Sendai virus fusogenic proteins HN and F. The vesicles were used as a vehicle to insert the PHA receptor glycoproteins into a highly enriched population of porcine B lymphocytes. Fluorescence analyses showed that 52 +/- 2% of the reconstituted B cells had incorporated the lectin receptors. The modified B lymphocytes were assayed for their response (tritiated thymidine incorporation into nucleic acids) to PHA, concanavalin A (Con A), or to lipopolysaccharide (LPS). The results showed that porcine B cells fused with vesicles containing only viral fusogenic proteins failed to respond to either PHA or Con A. Tritiated thymidine incorporation was similar to background values. The cells did, however, respond to LPS with values of label incorporation similar to those observed in the case of pre-fused B lymphocytes. When purified B lymphocytes were fused with vesicles containing PHA receptors and viral fusogenic proteins, assays of thymidine incorporation showed a statistically significant (P less than 0.001) and specific response of the modified cells to PHA stimulation. Reconstituted cells cultured in the presence of PHA incorporated approximately nine times more radioactive label than pre-fused cells or cells fused with vesicles containing only fusogenic viral proteins. In marked contrast, reconstituted B lymphocytes did not show any significant label incorporation above background level in response to Con A, but they retained their ability to respond to LPS. Our findings suggest that B lymphocytes can be made to respond specifically to PHA by insertion of appropriate lymphocyte-derived receptors.     

Immunoactivating potential of multilamellar liposome vesicles (MLV) in murine popliteal lymph node (PLN) test

Immunoactivating properties of subcutaneously injected small unilamellar vesicles (SUV) and multilamellar liposome vesicles (MLV) were studied in relation to different transition temperatures (Tc) of phospholipids. Liposome-induced proliferative reaction in the popliteal lymph node (PLN) was quantified by subsequent cytometric assay. Early cell activation during the onset of PLN reaction was monitored by immunophenotyping of lymphocyte subsets stained with a panel of monoclonal antibodies (mAbs) and gating the subset-specific large/activated cells. Injection of MLV liposomes containing distearoyl phosphatidylcholine (DSPC) and dipalmityl phosphatidylcholine (DPPC), characterized by relatively high Tc, resulted in a marked PLN reaction, increased numbers of CD4⁺, CD8⁺, Ig⁺ subsets and increased proportions of large/activated EAM⁺ (CD69⁺) and CD25⁺ (IL-2 receptor⁺) cells. The reaction was dose and time dependent. In contrast, injection of MLV liposomes containing lipids of low Tc, such as egg phosphatidylcholine (egg PC) and dimyristoyl phosphatidylcholine (DMPC), did not show any immunoactivation. In addition, there was a highly reduced immunoactivating potential of smallsize SUV liposomes over large-sized MLV of identical phospholipid composition. Generally, both lipid composition and vesicle size appeared to be essential for the immunoactivating potential of liposomes. The data suggest a possible correlation between the Tc of the phospholipid and the immunoactivating potential of the large-sized MLV liposomes.     

Plasma membrane functionalization using highly fusogenic immune activator liposomes

Cell surface functionalization and target molecule incorporation into living cell membranes without functional damage represent major biotechnological challenges. One possible way to achieve these goals is to induce cell membrane fusion with an artificial membrane containing molecules equipped with reactive groups or ligands. In this work we developed a carrier system to incorporate lipopolysaccharide (LPS), an immune cell activating molecule from Gram-negative bacteria, into mammalian membranes. LPS is not present in untreated mammalian cells which hence are not detectable by the immune system. Here, we demonstrate the successful incorporation of LPS into fusogenic liposomes (FLs) and subsequent incorporation into mammalian plasma membranes using these FLs. Additionally, the presence of LPS in cell membranes was probed by the addition of non-activated macrophages. A high concentration of LPS in the plasma membrane of immortalized fibroblasts activated the immune cells, which in turn started to eliminate LPS-exhibiting cells. Our method for cellular membrane functionalization is a promising tool for biomedical applications and could provide the basis for specific cell targeting approaches.     

Incorporation of recombinant gamma interferon into liposomes enhances its ability to induce peritoneal macrophage antitoxoplasma activity.

In this study we compared the ability of free- and liposome-incorporated murine recombinant gamma interferon (rIFN-gamma) to enhance peritoneal macrophage H2O2 release and antitoxoplasma activity in vitro. rIFN-gamma was efficiently (37 to 47%) incorporated into multilamellar vesicles composed of phosphatidylglycerol/cholesterol in a 2:1 molar ratio. The amount of rIFN-gamma incorporated into multilamellar vesicles and added to macrophages (0.1 to 1,000 U/ml) was quantitated with [3H]rIFN-gamma. The concentration of liposomal rIFN-gamma required to enhance macrophage H2O2 release (1 U/ml) and maximally inhibit Toxoplasma gondii growth (10 U/ml) was one-tenth the concentration required for free rIFN-gamma (10 and 100 U/ml, respectively). This increase in potency was observed in both thioglycolate-elicited and resident peritoneal macrophages. Control liposomes containing encapsulated buffer had no effect on the potency of free rIFN-gamma. The duration of macrophage activation induced by 24 h of liposomal rIFN-gamma treatment was also considerably longer than that induced by free rIFN-gamma (2 days versus less than 1 day). These data indicate that liposomal rIFN-gamma is more active than free rIFN-gamma as an inducer of macrophage microbicidal properties in vitro. This enhanced activity, combined with the potential for selective delivery of liposomal rIFN-gamma to phagocytic cells in vivo, may improve the therapeutic efficacy of rIFN-gamma in infections characterized by parasitization of phagocytes.     

A New Approach for the Immunogenic Presentation of Membrane-Bound Human Colon Tumor Antigens

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.     

A new approach for the immunogenic presentation of membrane-bound human colon tumor antigens

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.     

Quantitative reconstitution of isolated influenza haemagglutinin into liposomes by the detergent method and the immunogenicity of haemagglutinin liposomes.

The reconstitution of influenza virus haemagglutinin into liposomes from lipid/protein/detergent mixtures by detergent removal provides vesicles that are similar in structure to viral particles. The dissociation properties of haemagglutinin aggregates and the molar ratio of lipid to protein in the starting mixture are the key factors for the individual and total yield of protein incorporation into liposomes. Structural properties of the detergent used as well as special reconstitution conditions are of minor importance for the formation of haemagglutinin liposomes. As determined by radial immunodiffusion-, haemolysis- and fusion experiments, specific properties of haemagglutinin were maintained to a large extent on liposomal incorporation, but its immunogenicity is increased, if the antigen is incorporated into the lipid bilayer of liposomes.     

Detection of lytic antimycobacterial antibodies by immune lysis of liposomes sensitized to tuberculin

Two procedures were used in order to incorporate purified protein derivative tuberculin (PPD) from M. tuberculosis, strain H37Rv, into calcein-containing liposomes: formation of multilamellar vesicles (MLV) in a PPD solution or exposure of preformed MLV to this solution. Immune lysis of these PPD-sensitized MLV was studied in the presence of a hyperimmune anti-M. tuberculosis sheep serum using a specific pathogen-free rabbit serum as a source of complement. A 50% release of encapsulated calcein was observed spectrofluorometrically after 30 min and remained unchanged up to 2 h. The release of calcein in the absence of complement or of anti-H37Rv serum or by liposomes which did not contain PPD never exceeded 1-2%. Liposomes formed in PPD solution were more sensitive to anti-H37Rv serum than preformed liposomes exposed to PPD. Trials with human sera from ten tuberculous patients revealed the presence of specific lytic immunoglobulins. In the presence of sera from skin test negative, non-tuberculous subjects, calcein release was significantly lower. This opens the way to a new method for the study of the humoral immunity in tuberculosis.     

Protective effect of a muramyl dipeptide analog encapsulated in or mixed with liposomes against Candida albicans infection.

Encapsulation of N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine in multilamellar vesicles composed of phosphatidylcholine, cholesterol, and phosphatidylserine (7:6.7:3) or phosphatidylcholine and phosphatidylserine (7:3) reduced the amount of drug needed to protect against a Candida albicans intravenous infection. The 50% effective doses for encapsulated and free drug were 5.5 and greater than 80 mg/kg, respectively. The optimum treatment was twice (at days 4 and 2 preinfection) by the intravenous route. Intraperitoneal, subcutaneous, and oral routes of administration were ineffective. The same potentiation of anti-Candida activity was observed whether the lower dose of drug was encapsulated in multilamellar vesicles, mixed with multilamellar vesicles, or given either 1 h before or 1 h after multilamellar vesicles. It was postulated that the mechanism of action involved the retention of the liposomes by organs of the reticuloendothelial system, resulting in an enhanced response of the macrophages to the immunostimulating activity of the N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine given in conjunction with the vesicles.     

Preferential loss of DNA polymerase alpha following suppression of replicative DNA synthesis of guinea pig macrophages by the immunostimulants muramyl dipeptide or lipopolysaccharide

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate 3H-thymidine into trichloroacetic acid-insoluble fraction. In pulse-labeling experiments, the incorporated 3H-thymidine was detected in short fragments of DNA, which corresponded to the Okazaki fragments. These results indicate that the observed thymidine incorporation is due to nuclear DNA replication but not DNA repair. The observed DNA synthesis of the macrophages was remarkably suppressed when the cells were cultured in a presence of muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The significant decrease of DNA polymerase alpha activity was found in the cells treated with MDP or LPS. In contrast, the activity of polymerase beta was not at all affected by the same treatment.