PS Liposomes Research Articles

Phosphatidylserine in Diabetes Research.

Phospholipids are lipids that constitute the basic structure of cell membranes. In-depth research has shown that in addition to supporting cell structures, phospholipids participate in multiple cellular processes, including promoting cell signal transduction, guiding protein translocation, activating enzymatic activity, and eliminating dysfunctional/redundant organelles/cells. Diabetes is a chronic metabolic disease with a complicated etiology and pathology. Studies have shown that the level of certain phospholipids, for example, the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in liver tissue, is negatively associated with insulin sensitivity. In addition, PS is a phospholipid exhibiting extensive cellular functions in diabetes. For this review, we analyzed many PS studies focusing on diabetes and insulin sensitivity in recent years and found that PS participates in controlling insulin secretion, regulating insulin signaling transduction, and participating in the progression of diabetic complications by mediating coagulation disorders in the microvasculature or targeting mitochondria. Moreover, PS supplements in food and PS-containing liposomes have been shown to protect against type 1 and type 2 diabetes (T1D and T2D, respectively) in animal studies. Therefore, by summarizing the regulatory roles played by PS in diabetes and the potential of successfully using PS or PS-containing liposomes for diabetic therapy, we hope to provide new ideas for further research into the mechanisms of diabetes and for drug development for treating diabetes and its complications.

Involvement of splenic marginal zone macrophages in the recognition of systemically administered phosphatidylserine-coated liposomes in mice

Autoimmune diseases present a significant clinical problem, highlighting the need for the development of novel or improved therapeutic methods. One of the factors that causes autoimmune diseases is a defect in the clearance of apoptotic cells by phagocytes. Thus, improved apoptotic cell processing has been considered as a strategy to treat autoimmune diseases. However, therapeutic strategies focusing on apoptotic cell clearance have not been approved till date. We have reported that liposomes composed of phosphatidylserine (PS liposomes) exhibit anti-inflammatory or immunosuppressive effects in macrophages. A PS liposome display PS on its surface, which plays a crucial role in the phagocytosis of apoptotic cells by marginal zone macrophages (MZMs), a key player in the clearance of apoptotic cells, by recognizing PS exposed on the surface of apoptotic cells. Therefore, we hypothesized that PS liposomes could be used as “antigen delivery vesicles” to act as a substitute for apoptotic cells in the treatment of autoimmune diseases. In this study, we showed that systemically administered PS liposomes accumulated in the marginal zone of the spleen due to recognition of surface-displayed PS by MZMs because it was observed that liposomes without PS did not accumulate in the marginal zone. In conclusion, PS liposomes may be useful vehicles to function as active agents and/or antigens against autoimmune diseases.

Phosphatidylserine Receptor Enhancement of SARS-CoV-2 Entry: AXL as a Therapeutic Target for COVID-19

Abstract Phosphatidylserine (PS) receptors enhance infection of a wide range of enveloped RNA viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2. Under ACE2lo conditions, expression of members of the TIM and TAM families of PS receptors synergized with ACE2 to enhance SARS-CoV-2 infection; however, PS receptors alone did not mediate infection. PS receptors enhanced SARS-CoV-2 binding to the surface of cells. While PS receptors did not interact directly with purified SARS-CoV-2 spike, addition of PS liposomes reduced entry of VSV-SARS-CoV-2 spike pseudovirions, providing evidence that PS/PS receptor interactions are mediating the effect. AXL being abundant on airway cell lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of some lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. RNAseq studies in Vero E6 and A549-hACE2 cells demonstrated that bemcentinib reduced SARS-CoV-2 transcripts dramatically. The ability of bemcentinib to decrease coronavirus infection in vivo was assessed using the betacoronavirus mouse hepatitis virus (MHV). Liver titers and virus load of MHV were significantly inhibited by bemcentinib at day 5 of infection. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and that inhibition of signaling of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

T202. THE EFFECT OF ANTIPSYCHOTIC DRUGS ON MEMBRANE FUSION: AN IN VITRO STUDY

BackgroundCommon clinical use of antipsychotics (AP) drugs shows that their therapeutic mode of action still needs further clarification although it is admitted that the Dopamine receptor D2 (D2R) antagonism plays a significant role. For instance, clozapine (CLOZ) - which is known to be the most effective AP in treating schizophrenic symptoms - has strikingly the lowest D2R antagonism. Non direct receptor-related effects might thus be involved in the activity of AP at the synapse level.AP, as well as neurotransmitters, are mostly lipophilic and insert within membranes. This characteristic is of interest as a significant proportion of schizophrenic patients has specific and abnormal membrane lipid composition. This possible proxy of the disease biotype can participate in the disease’s physiopathology but also be critical for the effect of AP drugs.We hypothesize that AP insertion into lipid membranes also contribute to their therapeutic effect. AP-induced modifications of synaptic membranes biophysics are likely to influence neurotransmission. In this study, we focus on the effect of AP on membrane fusion, a crucial step for the exocytosis of neurotransmitters.MethodsLiposomes modelling synaptic vesicles were reconstituted in saline buffer. Two standard ternary and quaternary lipid mixtures have been studied: phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine (PC:PE:PS) [65:25:10] and the synaptic-like PC:PE:PS:sphingomyelin:cholesterol (PC:PE:PS:SM:CHOL) [25:25:10:10:30]. Some liposomes were protein-free and others were functionalized with Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) proteins, which trigger in vivo the fusion of synaptic vesicles with the pre-synaptic plasma membrane. The liposome size was checked by Dynamic Light Scattering. Insertion of AP within the membrane was checked by second derivative spectroscopy. Fusion was measured by Fluorescence Resonance Energy Transfer in the absence or presence of CLOZ or chlorpromazine (CPZ) at various lipid:AP ratios (10:1 to 100000:1). Protein-free liposomes were fused with Polyethylene glycol (PEG) and SNARE liposomes through the action of cognate SNARE proteins residing in their membrane.ResultsLiposomes of the same lipid composition were of the same size, with no effect of the addition of AP drugs at various concentrations. Molar partition coefficient of AP drugs within the membrane of protein-free liposomes was approximately 70–85%. CPZ or CLOZ inhibited the fusion of PC:PE:PS liposomes by about 20–40%. When liposomes were synaptic-like (PC:PE:PS:SM:CHOL), the inhibition of fusion by AP drugs reached 50%. CLOZ also inhibited SNARE-mediated fusion of PC:PE:PS liposomes by about 30%. This effect on SNARE-mediated fusion was not observed with CPZ.DiscussionAltogether, these results, despite preliminary, could help to understand partially a non direct receptor-related effect of antipsychotics. Indeed, these drugs also seem to modify membrane dynamics at the synapse level. This seems to be particularly the case of CLOZ on SNARE-mediated fusion and could explain its specific therapeutic efficiency.

Peptidomimetic inhibitors targeting the membrane-binding site of the neutrophil proteinase 3

Proteinase 3 (PR3), together with other serine proteases, such as neutrophil elastase (NE) and cathepsin G (CG), regulates inflammatory and immune responses. However, in comparison with NE and CG, there is increasing evidence that PR3 functions significantly differ. In particular, PR3 can bind to cell membranes and such membrane-bound PR3 (mbPR3) might be differently involved in the activation of cytokines, growth factors, cellular receptors, and in the regulation of cell apoptosis. For instance, PR3 membrane binding can block some “eat me” signals, notably, phosphatidylserine membrane lipid, and facilitate non-resolving inflammation. Based on the clear evidence that PR3 membrane binding affects the biological functions of PR3, we designed peptidomimetic inhibitors that can remove mbPR3 from the membrane surface in vitro without influencing PR3 catalytic activity. Such inhibitors, which specifically target PR3 binding to membranes, are still lacking. In particular, we found peptidomimetics that inhibit binding of PR3 to POPC:PS liposomes, which mimic the biological environment of PR3.

Abstract B119: Characterization of a phosphatidylserine, TAM receptor (Tyro3, Axl, Mertk), PDL1 axis in breast cancer

Abstract Tyro3, Axl and Mer (TAMs) are three homologous receptor tyrosine kinases that bind vitamin K-dependent endogenous ligands, Protein S (PROS1) and Gas6, and act as bridging molecules to promote PS-mediated clearance of apoptotic cells (efferocytosis). In recent years, evidence has accumulated that TAMs are overexpressed in a wide array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo and radio resistance to targeted therapeutics. TAMs have also been implicated as inhibitory receptors on myeloid-derived cells that suppress anti-tumor immunity. In addition to TAM overexpression, externalized PS is also concomitantly up-regulated in the tumor microenvironment, suggesting that PS and PS receptors might act as immune checkpoint inhibitors. To better understand the biology of TAMs, and the specificity of interaction between TAMs and PS, we generated chimeric TAM reporter cell lines comprised of the extracellular domains of each TAM fused to the intracellular domains of the IFNR1, as well as cell lines stably expressing full-length native TAMs. Using these systems, we found that each TAM receptor has a unique pattern of activation by GAS6 and PROS1, as well as unique dependency for PS on apoptotic cells, PS liposomes, and exosomes. Interrogating epithelial cells that express WT TAMs, we also observed that each TAM showed differential capacity for efferocytosis, AKT-mediated chemo-resistance, and their ability to up-regulate the immune checkpoint inhibitory ligand PD-L1 on breast cancer cells. Functionally, TAM-mediated efferocytosis and PD-L1 up-regulation could be partially blocked by PS targeted antibodies 11.31 and bavituximab, suggesting the existence of a PS/PS-R (TAM-receptor)/PD-L1 axis that drives immune escape in solid cancers. These studies demonstrate that, despite their similarities, Tyro3, Axl and Mertk are likely to perform distinct functions in both immune-regulation and the recognition and removal of apoptotic cells. Finally, these studies provide a rationale for combinatorial therapeutics targeting PS, TAM, and PD-L1 as immune checkpoint inhibitors in the treatment of cancer. Citation Format: Canan Kasikara, Sushil Kumar, Ke Geng, Viral Davre, Cyril Empig, Bruce Freimark, Michael Gray, Kyle Schlunegger, Jeff Hutchins, Sergei V. Kotenko, Raymond B. Birge. Characterization of a phosphatidylserine, TAM receptor (Tyro3, Axl, Mertk), PDL1 axis in breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B119.

Abstract 549: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase

Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that acidic pH exposes apoAI’s hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation. Methods and Results: We found that apoAI unfolding in guanidine was facilitated at acidic pH (EC 50 for unfolding at 0.94 and 0.75 M guanidine at pH 7.5 and 5, respectively), and confirmed that acidic conditions promoted apoAI solubilization of and binding to egg PC:PS (8:2 mole ratio) liposomes. We generated a FITC/Alexa647-labeled fluorescent ratiometric apoAI pH indicator whose FITC/Alexa647 emission ratio decreased as pH decreased. Using flow cytometry in BHK and RAW264.7 cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37 o C led to substantial acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21 o C or with an inhibitor of endocytosis, where apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V 1 domain and an embedded V 0 domain. We observed that bafilomycin, the V-ATPase inhibitor, eliminated the pH shift of apoAI on plasma membrane, and dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI. Although ABCA1 did not alter total cellular levels of V-ATPase, we showed that ABCA1 increased the cell surface levels of the V 0 A1 and V 1 E1 subunits of V-ATPase by cell surface biotinylation and immunofluorescence. Moreover, inhibition of apoAI acidification by bafilomycin decreased apoAI unfolding on the cell surface. Conclusions: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which promotes apoAI unfolding and lipidation.

Abstract 323: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase

Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that apoAI can solubilize liposomes made with egg PC:PS (8:2) only at acidic pH, where apoAI has unfolded to expose more hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation. Methods and Results: We found that apoAI unfolding in guanidine was facilitated at pH 5.0 vs. pH 7.5 (EC50 = 0.75 M and 0.94 M guanidine, respectively). We confirmed that apoAI solubilization of and binding to POPC:PS liposomes was only effective at pH < 5.5. We then designed a fluorescent ratiometric apoAI pH indicator labeling apoAI with FITC and Alexa647, and validated it in solution assays showing that the FITC/Alexa647 ratio decreased as the pH decreased. Using flow cytometry in both RAW264.7 and BHK cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37 o C led to a large acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21 o C or with an inhibitor of endocytosis, where the vast majority of apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V 1 domain and an embedded V 0 domain. We observed that bafilomycin, the V-ATPase inhibitor, dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI, and also eliminated the pH shift of apoAI on plasma membrane. We showed V-ATPase was expressed on the cell surface by cell surface biotinylation. Although the total level of V 0 A1 subunit didn’t change in ABCA1 expressing cells, ABCA1 induced the cell surface level of the V 0 A1 subunit. Conclusion: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which may promote apoAI unfolding and lipidation.

Preparation and Characterization of Pulmonary Surfactant-Super Oxide Dismutase Liposomes

Objective: To prepare pulmonary surfactant-superoxide dismutase (PS-SOD) Liposomes and analyze the biological characteristics of them. Methods: To prepare PS-SOD Liposomes with rotary evaporation method and analyze the formulation and bioactivity of them, including shape, size, encapsulating ratio, antioxidant activity and surface tension. Results: The PS-SOD Liposome is a kind of ivory white suspension and presents to be a fairly homogeneous spherical article with a mean grain size of 0.463±0.223μm. There is no change in antioxidant activity before and after SOD being encapsulated to be PS-SOD Liposomes. No difference has been found in surface tension between PS and PS-SOD Liposomes. Conclusion: PS-SOD Liposomes can be successfully prepared through rotary evaporation method and maintain stable biological activities both of PS and SOD. Source of financial support: National Natural Science Foundation of China, NO.30471714;

Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo

Phosphatidylserine (PS) on apoptotic cells promotes their uptake and induces anti-inflammatory responses in phagocytes, including TGF-beta release. Little is known regarding the effects of PS on adaptive immune responses. We therefore investigated the effects of PS-containing liposomes on immune responses in mice in vivo. PS liposomes specifically inhibited responses to Ags as determined by decreased draining lymph node tissue mass, with reduced numbers of total leukocytes and Ag-specific CD4(+) T cells. There was also a decrease in formation and size of germinal centers in spleen and lymph nodes, accompanied by decreased levels of Ag-specific IgG in blood. Many of these effects were mimicked by an agonistic Ab-specific for the PS receptor. TGF-beta appears to play a critical role in this inhibition, as the inhibitory effects of PS were reversed by in vivo administration of anti-TGF-beta Ab. PS-containing liposomes did not appear to directly inhibit dendritic cell maturation in vitro in response to a variety of stimuli, nor did it prevent their migration to regional lymph nodes in vivo, suggesting that the inhibitory effects may have resulted from complicated interactions between tissue cells and dendritic cells, subsequently inhibiting their ability to productively activate T lymphocytes.

Phosphatidylserine regulates the maturation of human dendritic cells.

Phosphatidylserine (PS), which is exposed on the surface of apoptotic cells, has been implicated in immune regulation. However, the effects of PS on the maturation and function of dendritic cells (DCs), which play a central role in both immune activation and regulation, have not been described. Large unilamellar liposomes containing PS or phosphatidylcholine were used to model the plasma membrane phospholipid composition of apoptotic and live cells, respectively. PS liposomes inhibited the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83, as well as the production of IL-12p70 by human DCs in response to LPS. PS did not affect DC viability directly but predisposed DCs to apoptosis in response to LPS. DCs exposed to PS had diminished capacity to stimulate allogeneic T cell proliferation and to activate IFN-gamma-producing CD4(+) T cells. Exogenous IL-12 restored IFN-gamma production by CD4(+) T cells. Furthermore, activated CTLs proliferated poorly to cognate Ag presented by DCs exposed to PS. Our findings suggest that PS exposure provides a sufficient signal to inhibit DC maturation and to modulate adaptive immune responses.

Plasma membrane cholesterol controls the cytotoxicity of Alzheimer's disease AbetaP (1-40) and (1-42) peptides.

Cell degeneration in Alzheimer's disease is mediated by a toxic mechanism that involves interaction of the AbetaP peptide with the plasma membrane of the target cell. We report here that PC12 cells become resistant to the cytotoxic action of AbetaP when incubated in a medium that enriches cholesterol levels of the surface membrane. On the other hand, making cholesterol-deficient membranes by either cholesterol extraction with cyclodextrin or by inhibiting de novo synthesis of cholesterol makes PC12 cells more vulnerable to the action of AbetaP. Increasing cholesterol content of PS liposomes also suppresses AbetaP-dependent liposome aggregation. We suggest that by modifying the fluidity of the neuronal membranes, cholesterol modulates the incorporation and pore formation of AbetaP into cell membranes. This idea is supported by our finding that the enhanced cytotoxicity generated by lowering the membrane cholesterol content can be reversed by AbetaP calcium channel blockers Zn2+ and tromethamine.

The effect of lipid molecular packing stress on cationic liposome-induced rabbit erythrocyte fusion

The effect of curvature stress on the efficiency of cationic liposome-induced fusion between rabbit erythrocytes was studied. Multilamellar cationic liposomes containing 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and different PEs (1,2-dilinoleoyl- sn-glycero-3-phosphoethanolamine (dilin-PE), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE), and lysophosphatidylethanolamine, egg (lyso-PE)) were used to induce cell–cell fusion. It was found that high cell–cell fusion yield (FY) of about 50% could be achieved in sucrose solution by using cationic liposomes containing 50% DOTAP. Cell–cell fusion was assayed by shape criterion and was verified by fluorescence microscopy, using a membrane dye mixing method. The curvature stress, as a result of mixing unsaturated PEs in cationic liposomes, had a significant effect on cell–cell FY which increased with the degree of acyl chain unsaturation, in the order dilin-PE>DOPE>POPE>lyso-PE. Replacement of dilin-PE, DOPE, or POPE by lyso-PE gradually in cationic liposomes lowered the cell–cell FY from 50% to 1%. Furthermore, cationic liposome-induced cell lysis, and fusion between cationic liposomes and cells, as assayed by the N-(lissamine rhodamine B sulfonyl)-1,2-dihexadecanoyl- sn-glycero-3-phosphoethanolamine, triethylammonium salt and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rh-PE/NBD-PE) energy transfer method, followed the same order as that for cell–cell fusion. Fusion between the negatively charged PS liposomes and cationic liposomes also followed the same order. The electric double layer screening by electrolytes in NaCl-containing solution and phosphate buffered saline (PBS) was found to reduce the cell–cell FY and cell lysis. These findings suggest that liposome-induced cell–cell fusion was achieved by cationic liposomes serving as fusion-bridges among cells.

Interaction of annexins IV and VI with phosphatidylserine in the presence of Ca2+: monolayer and proteolytic study.

Annexins, Ca2+- and phospholipid-binding proteins are known to bind to artificial and biological membranes in a calcium-dependent manner. However, the precise mechanism of the annexin-membrane interactions still remains to be studied in detail. In this paper we describe the results of studies on the interactions of the annexin/Ca complexes with phospholipids, obtained by the Wilhelmy balance method of assessing the surface pressure of a phospholipid monolayer. We show that the annexin IV/Ca as well as annexin VI/Ca complexes significantly reduce the surface pressure of a phosphatidylserine monolayer, when its initial value is close to collapse pressure. The effect is highly specific for monolayers composed of phosphatidylserine and strongly sensitive to pH and ionic strength. The most pronounced changes have been observed at pH 7.0-7.5, at a protein/Ca molar ratio of 1:2 for annexin IV and 1:4 for annexin VI. In the presence of sodium chloride at concentrations exceeding 400mM this effect was almost completely abolished. The obtained results point to the mainly electrostatic character of the annexin/phosphatidylserine interactions. In addition, using large multilamellar lipid vesicles and serine proteases, we demonstrate that annexins, when bound in a ternary complex with phospholipids and calcium ions, are partially protected against proteolysis. Our observation that annexin molecules, complexed with calcium ions, are protected against proteolytic attack in the presence of PS liposomes does not have to be necessarily explained in terms of partial penetration of protein within the membrane bilayer.

Vesicle Aggregation by Annexin I: Role of a Secondary Membrane Binding Site

Proteins of the annexin family bind to and aggregate secretion granules or liposomes in the presence of Ca2+. We investigated the mechanism of vesicle aggregation performing experiments in which annexin I bound to PS liposomes was allowed to aggregate additional liposomes. The protein was initially bound to PS liposomes in 50-100 microM Ca2+ under nonaggregating conditions; then further liposomes were added, and aggregation was started by increasing Ca2+ to 0.5-1 mM. Coaggregation between both liposome populations was followed using resonance energy transfer (RET) and turbidimetric techniques. In RET experiments, annexin I was bound to liposomes containing N-(7-nitro-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE), and the second liposome population contained lissamine-Rhodamine-PS. Coaggregation was estimated from NBD fluorescence quenching. Rapid fluorescence and turbidimetric changes were observed, demonstrating coaggregation between both populations of liposomes. Therefore, annexin I molecules may bind two membranes in a bivalent fashion. Rates of coaggregation were similar to the rates of aggregation observed when all vesicles contained protein, indicating that aggregation is mediated only by bivalent annexin I molecules. Thus, membrane aggregation is due to a secondary membrane binding site in annexin I. PS liposomes containing annexin I coaggregated with phosphatidylcholine (PC) liposomes, demonstrating that membrane-bound annexin I binds PC, in contrast with soluble annexin I. Secondary binding to PC was significantly slower than secondary binding to PS, pointing to the importance of negative charge in the secondary membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

PC:PS liposomes induce a recruitment of neutrophils and the release of TNF alpha in the lungs of mice sensitized with Saccharopolyspora rectivirgula.

The aim of this study was to verify the effect of nasally instilled liposomes (L) or dexamethasone-containing L (Ldexa) on normal or inflamed lung tissue. Three groups of mice were studied. Group I was given saline instillations for 3 weeks prior to the instillation with liposomes. In groups II and III lung inflammation was induced by repeated instillations of Saccharopolyspora rectivirgula before the instillation of liposomes (group II) or liposomes containing dexamethasone (group III). Animals from all groups were killed at regular time intervals for up to 48 h after the instillation of liposomes. The total cell count in bronchoalveolar lavage fluid did not differ significantly between groups I and II. However, in group III it decreased rapidly from 6.2 to 2.8 x 10(5) cells mL-1 within 2 h. Differential counts did not change in group I, but in group II a transient neutrophilia was observed 180 min after the instillation of liposomes. In group III, the instillation of dexamethasone-containing liposomes depleted all neutrophils and lymphocytes after 4 h. No TNF alpha was found in samples of lavage fluid from any of the groups at time 0. In group I, liposomes induced the production of 0.03 ng mL-1 of TNF alpha in the 1 h sample only. In group II, TNF alpha peaked to 1 ng mL-1 at 1 h and had decreased to 0.35 ng mL-1 by 3 h. In group III, TNF alpha peaked at 1 h, but only reached a level of 0.1 ng mL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Granuloma formation in mice by liposomes of new glycolipid containing mycolic acids, "trehalose 2,3,6'-trimycolate"

After intravenous administration of liposomes containing a novel glycolipid, trehalose 2,3,6'-trimycolate, isolated from Rhodococcus aurantiacus, the granuloma formation was observed distinctively in the lungs, spleen and liver of ICR mice. The concentration of the glycolipid in liposomes influenced profoundly on the inducibility for granulomas and the liposomes containing 10 mol% of trehalose 2,3,6'-trimycolate showed the highest activity for the granuloma formation in mice without a significant loss of body weight of mice with a less toxicity. EggPC liposomes are of more highly inducible for the granuloma formation in mice than eggPC:PS (7:3, mol ratio) liposomes or eggPC:Chol (9:1, mol ratio) liposomes, suggesting that cholesterol in liposomes was not necessarily essential for inducing granulomas in mice. Among the various PC liposomes, synthetic dimyristoyl-phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes were of highly inducible, although a high responsiveness was also observed with natural lecithines such as egg or soy PC. However, distearoylphosphatidylcholine (DSPC) liposomes did not show any activity for the granuloma formation in any organs of mice. Organ responsiveness differed significantly in the micelle forms injected. Glycolipid entrapped with eggPC-Chol liposomes showed a lower lung index below 1.0, in contrast to w/o/w micelles containing Freunds' incomplete adjuvant, which showed a higher granuloma formation in the lungs. It was also noted that the toxicity of glycolipid containing liposomes was comparatively lower than the toxicity of glycolipid containing w/o/w micelles, indicating that the liposomes appeared to be more suitable for the induction of the immunomodifying activities of mycolic acid-containing glycolipids than w/o/w micelles.

Calcium phosphate precipitation in aqueous suspensions of phosphatidylserine-containing anionic liposomes.

Liposomes prepared from 6.3:1.8:0.9:1.0 molar mixtures of phosphatidylcholine, dicetyl phosphate, cholesterol, and phosphatidylserine, respectively, (PS(+) liposomes) were compared with similarly prepared liposomes without the phosphatidylserine (PS(-) liposomes) for their effect on calcium phosphate precipitate formation in aqueous solutions at pH 7.4 and 22 degrees C. The liposomes, encapsulated with 50 mM phosphate (PI), were suspended in buffered 2.2 mM CaCl2, 0 or 1.5 mM KH2PO4 solutions and made permeable to Ca2+ fluxes with the ionophore, X-537A. External solution Ca2+ losses were found to be small in both PS(+) and PS(-) liposome suspensions when no ionophore was added. Even with 1.5 mM PI in the external solution, these losses did not exceed 0.2 mM. However, inoculating both liposome preparations with X-537A resulted in rapid, appreciable losses in solution Ca2+. Previous studies showed that in PS(-) liposomes, these latter losses were due to calcium phosphate precipitation, with the precipitate confined to the interior of the liposomes when no external PI was present, but extending to outside the liposomes when the suspending medium was rendered metastable. In the present study, Ca2+ losses resulting from intraliposomally confined precipitation were found to be marginally greater in PS(+) liposomes due primarily to a larger volume of entrapped PI available for reaction in these liposomes. However, with the addition of PI to the external solution, the reverse was observed, i.e., considerably less Ca2+ was lost in PS(+) than in PS(-) suspensions, a result of markedly less X-537A-induced precipitate forming outside PS(+) liposomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase of globin RNA synthesis induced by phosphatidylserine liposomes in isolated erythroleukemic cell nuclei. Morphological and functional features.

The effect of phospholipid vesicles on chromatin structure, protein composition and globin RNA synthesis has been analysed in nuclei isolated from murine erythroleukemia cells. In terms of chromatin organisation, PC vesicles with neutral surface charge do not affect the structure of chromatin fibres, whereas negatively charged PS vesicles induce chromatin decondensation to a great extent. Indeed the fibres appear uniformly dispersed lacking also the perinucleolar heterochromatin. These morphological features are accompanied by depletion of lysine-rich histones H1 and H1(0) and of histone-like protein A5, due to PS liposomes. Functionally, PS vesicles induce enhancement both of total RNA and specific mRNA synthesis, as analysed by in vitro transcription of beta globin gene. On the contrary, PC vesicles do not seem to affect significantly total RNA and globin mRNA synthesis. These observations fit well with previous data obtained in other experimental systems, and support both the use of these molecules as probes for chromatin structure and function and their possible involvement in transcriptional events.

Disposition of intact liposomes of different compositions and of liposomal degradation products

Small unilamellar liposomes containing bovine serum albumin were prepared by a new double-emulsion technique and administered to mice and rats in intravenous injections. The elimination of intact liposomes, the association of phospholipid marker with lipoproteins, and the appearance of released internal marker and its degradation products were followed by column chromatography of plasma samples. In vitro labelled lipoproteins were administered to the animals in intravenous injections together with free bovine serum albumin and the elimination of the two substances was studied by closely related techniques. The clearance of intact PC : PS (4 : 1) liposomes from plasma was biphasic and much faster than that of labelled lipoproteins and bovine serum albumin either originating from liposomes or injected as such. The second elimination phase for these liposomes was barely detectable by our analytical methods. In contrast, DSPC : CHOL (2 : 1) liposomes showed a very significant second-phase elimination, with a half-life of 12 hr for the intact liposomes. In tissue distribution studies in mice, the major accumulation of liposomal markers was found in the liver and spleen, and less in the kidneys and intestinal wall. Uptake into liver and spleen appeared to be due to the uptake of intact liposomes, whereas the uptake into kidneys and gut wall was caused by the uptake of liposomal degradation products. The uptake of PC : PS (4 : 1) liposomes into the liver was higher than that of DSPC : CHOL (2 : 1) liposomes; the opposite was the case with their uptake into the spleen. In rats, too, liposomes of different compositions showed significant variations in stability and in plasma half-lives of intact liposomes. Generally, there was a considerable increase in the liposomal stability in the presence of cholesterol and when use was made of a phospholipid with a high transition temperature.