Phosphatidylserine In Liposomes Research Articles

The Peroxisomal Localization of Hsd17b4 Is Regulated by Its Interaction with Phosphatidylserine.

Phosphatidylserine (PS), a negatively charged phospholipid exclusively located in the inner leaflet of the plasma membrane, is involved in various cellular processes such as blood coagulation, myoblast fusion, mammalian fertilization, and clearance of apoptotic cells. Proteins that specifically interact with PS must be identified to comprehensively understand the cellular processes involving PS. However, only a limited number of proteins are known to associate with PS. To identify PS-associating proteins, we performed a pulldown assay using streptavidin-coated magnetic beads on which biotin-linked PS was immobilized. Using this approach, we identified Hsd17b4, a peroxisomal protein, as a PS-associating protein. Hsd17b4 strongly associated with PS, but not with phosphatidylcholine or sphingomyelin, and the Scp-2-like domain of Hsd17b4 was responsible for this association. The association was disrupted by PS in liposomes, but not by free PS or the components of PS. In addition, translocation of PS to the outer leaflet of the plasma membrane enriched Hsd17b4 in peroxisomes. Collectively, this study suggests an unexpected role of PS as a regulator of the subcellular localization of Hsd17b4.

Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.

Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.Supplementary informationThe online version of this article (doi:10.1038/mi.2015.137) contains supplementary material, which is available to authorized users.

An Increased Adjuvanticity of Liposomes by the Inclusion of Phosphatidylserine in Immunization with Surface-Coupled Liposomal Antigen

Background: Exposure of phosphatidylserine (PS) on apoptotic cells is known to result in the enhanced recognition of apoptotic cells by phagocytes. By the inclusion of PS in the lipid component of liposomes, increased liposome immune adjuvant activity was expected. Methods: In the present study, two different liposome preparations containing either PS, i.e. PS-liposome, or phosphatidylcholine (PC), i.e. PC-liposome, were made, and macrophage recognition, processing, and antigen presentation of surface-coupled liposomal antigen were compared. Results: When ovalbumin-liposome conjugates were added to a culture of macrophages, enhanced recognition and processing of ovalbumin by the macrophages were observed by the inclusion of PS in the liposomes. The results correlated well with those regarding macrophage antigen presentation of liposome-coupled ovalbumin. Furthermore, in vivo immunization in mice with ovalbumin-liposome conjugates made with PS-liposomes induced a significantly higher level of anti-ovalbumin IgG antibody production than was induced by ovalbumin-liposome conjugates made with PC-liposomes. IgE-selective unresponsiveness was induced by ovalbumin-liposome conjugates regardless of the lipid components of liposomes. Conclusions: These results suggest that the inclusion of PS in liposomes enhances recognition and processing of surface-coupled liposomal antigen by macrophages, and increases liposome immune adjuvant activity.

Characterization of calcitonin-containing liposome formulations for intranasal delivery

Calcitonin-containing liposome formulations were characterized to obtain information for evaluation of their feasibility in intranasal delivery. The parameters of liposomal charge characteristics, charge inducing agent concentration, calcitonin concentration and pH of the medium on the loading efficiency and leakage behaviour, and the chemical stability of calcitonin in liposomes were investigated. Results showed that the loading efficiency of calcitonin increased with increasing the added concentration of calcitonin. The magnitude of the loading efficiency due to the liposomal charge of negative, positive and neutral characteristics was in the order of negatively charged liposome > neutral liposome > positively charge liposome. The increase of molar ratio of phosphatidylserine in liposomes showed an increase of loading efficiency; while, the increase of molar ratios of stearylamine showed a decrease of loading efficiency. The loading efficiency at pH7.4 was greater than that at pH4.3. The leakage of positively charged liposomes was greater than that of neutral and negatively charged liposomes. The leakage at pH4.3 was faster than that at pH7.4. The leakage of positively charged liposomes increased as temperature increased. The chemical stability of calcitonin in both solution and liposomes demonstrated a pseudo-first-order kinetic degradation. Less degradation was observed at pH3.4 and 4°C. The degradation rate of calcitonin in solution, or in positively charged, negatively charged, and neutral liposomes, exhibited no significant difference. The particle size of the calcitonin-containing liposomes after storage for 1 month at pH4.3 and 4°C showed little change.

Uptake of Liposomes Containing Phosphatidylserine by Liver Cellsin Vivoand by Sinusoidal Liver Cells in Primary Culture:In Vivo–in VitroDifferences

The interaction with liver cells of liposomes containing different mol fractions of phosphatidylserine was investigatedin vivoandin vitro.Increasing the amount of liposomal phosphatidylserine from 10 to 30 mol% leads to a faster blood disappearance of the liposomes. Within the liver, which is mainly responsible for this elimination, these liposomes are only taken up by the hepatocytes and Kupffer cells. By contrast, sinusoidal endothelial cells,in vitro,do bind and internalize liposomes containing ≥30% phosphatidylserine at least as actively as Kupffer cells. The uptake by endothelial and Kupffer cells is inhibited by poly(inosinic acid) and other anionic macromolecules, suggesting the involvement of scavenger receptors. The lack of liposome uptake by endothelial cells underin vivoconditions can be attributed to plasma effects since addition of various sera caused severe reduction ofin vitrouptake of liposomes.In vivothe phosphatidylserine head groups may be masked by plasma proteins adsorbed to the liposomal surface, thus preventing recognition by receptors, which are intrinsically able to recognize phosphatidylserine.

Liposomal phosphatidylserine inhibits tumor cytotoxicity of liver macrophages induced by muramyl dipeptide and lipopolysaccharide

Liposomes can very efficiently deliver immunomodulators to macrophages so as to induce tumor cytotoxicity. Liposomes most widely used for that purpose contain negatively charged lipids, in particular phosphatidylserine (PS), to enhance liposome uptake by the macrophages. We investigated the effect of three negatively charged liposomal lipids on the in vitro activation of liver macrophages to tumor cytotoxicity by muramyl dipeptide (MDP) and lipopolysaccharide (LPS). Both MDP- and LPS-induced tumor cytotoxicity towards murine colon adenocarcinoma cells were strongly inhibited by PS-containing liposomes. Under comparable conditions phosphatidylglycerol (DPPG)-containing or dicetyl phosphate (DCP)-containing liposomes did not inhibit or only marginally inhibited the induction of tumor cytotoxicity. We did not observe PS-mediated inhibition of tumor cell toxicity when the exposure of the macrophages to PS-liposomes was limited to the 4-h activation period prior to addition of the tumor target cells, suggesting that the inhibitory effect is accomplished at the level of the later stages of the activation process. Previously, we showed that macrophages which are activated to tumor cytotoxicity during a 24-h incubation with MDP become refractory to a second activation with MDP. Now we observed that simultaneous incubation with PS-containing liposomes partially prevents this refractoriness, which is also compatible with an interfering action of PS at a relatively late stage in the activation process. We conclude that PS, despite its reported stimulatory effect on liposome uptake by macrophages, can seriously antagonize the effectiveness of immunomodulating agents acting on macrophages. This bears relevance to the use of PS-containing liposomes as a vehicle for such agents. The results are discussed in perspective of earlier reported pharmacological effects of PS and its metabolites.

Interaction of bovine heart pyruvate kinase with phospholipids

The interaction between bovine heart pyruvate kinase and liposomes was investigated for various phospholipids as function of pH, and salt concentration using steady-state kinetics and ultracentrifugation. Liposomes made from erythrocyte total lipid fraction and individual phospholipids were used. Pyruvate kinase specific activity increases upon the interaction with the phospholipids. The activation is specifically sensitive to presence of phosphatidylserine in liposomes. l-serine, and phospho- l-serine which are main components of phosphatidylserine head group show also some activation effect. Efficient adsorption of pyruvate kinase to phosphatidylserine liposomes occurs in the pH range 6.0–8.0 and at low ionic strength. Interaction with phosphatidylserine liposomes results in the change of V max d and K m values for phospho enolpyruvate without marked effect on K m value for ADP, and Hill coefficients for both substrates. e The interaction does not seem to influence the cooperativity between binding sites.

Quantitative Determination of Phosphatidylethanolamine and Phosphatidylserine in Liposomes and on HPTLC Plates with o-Phthalaldehyde

Quantitative Determination of Phosphatidylethanolamine and Phosphatidylserine in Liposomes and on HPTLC Plates with o-Phthalaldehyde

Transport and decar☐ylation of liposomal phosphatidylserine: effect of cations

Decar☐ylation of liposomal phosphatidylserine by rat liver and Ehrlich ascites tumor mitochondria was taken as a measure of phospholipid transfer. The process was found to be greatly enhanced by the cytoplasmic fraction of rat liver containing nonspecific lipid transfer protein, but not by the cytoplasmic fraction from tumor cells. Divalent cations, like rat liver cytoplasmic fraction, also stimulated phosphatidylserine decar☐ylation by facilitating the lipid association with mitochondria. In contrast, these cations, at 0.5–3 mM concentration, inhibited the cytoplasmic fraction-mediated phosphatidylserine transport. Monovalent cations were also inhibitory but at 20–150 mM concentration. However, they had no effect on phosphatidylserine decar☐ylation in the absence of the cytoplasmic fraction. Further experiments with purified rat liver nonspecific lipid transfer protein and pyrene-labeled phosphatidylcholine and phosphatidylserine have shown that cations by neutralizing net negative charge on phospholipid donor vesicles decrease the interaction of protein with them and, in consequence, lower the rate of release of molecules to the water phase.

Antibodies to liposomal phosphatidylserine and phosphatidic acid

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.

Reconstitution of phosphatidylserine import into rat liver mitochondria

The synthesis translocation and decarboxylation of phosphatidylserine occurs in a cell-free system. The principal membrane components necessary are microsomes (source of phosphatidylserine synthase) and mitochondria (source of phosphatidylserine decarboxylase). The interorganelle translocation of phosphatidylserine can be measured by quantitating the decarboxylation of phosphatidyl[1'-14C]serine initially present in prelabeled microsomal membranes using a 14CO2 trapping assay. The decarboxylation of microsomal phosphatidylserine by intact mitochondria is 1) dependent upon substrate (microsomal membrane) concentration, 2) different from decarboxylation of liposomal phosphatidylserine, 3) resistant to proteases, 4) independent of soluble factors, and 5) unaffected by the addition of partially purified phospholipid exchange proteins but accelerated by purified nonspecific phospholipid exchange protein. The rate-limiting step in the reconstituted translocation-decarboxylation system is not the decarboxylation reaction but the initial translocation event between the microsomal membrane and the outer mitochondrial membrane. These data are interpreted to demonstrate that phosphatidylserine import into the mitochondria can occur via collision complexes formed between the endoplasmic reticulum or vesicles derived therefrom and the outer mitochondrial membrane.

The intrinsic pKa values for phosphatidylserine and phosphatidylethanolamine in phosphatidylcholine host bilayers

Potentiometric titrations and surface potential measurements have been used to determine the intrinsic pKa values of both the carboxyl and amino groups of phosphatidylserine (PS) in mixed vesicles of PS and phosphatidylcholine (PC), and also of the amino group of phosphatidylethanolamine (PE) in mixed PE-PC vesicles. The pKa of the carboxyl group of PS in liposomes with different PS/PC lipid ratios measured by the two different methods is 3.6 +/- 0.1, and the pKa of its amino group is 9.8 +/- 0.1. The pKa of the amino group of PE in PE-PC vesicles, determined solely by surface potential measurements, is 9.6 +/- 0.1. These pKa values are independent of the aqueous phase ionic strength and of the effect of the liposome's surface potential due to the presence of these partially charged lipids.

Conformational changes of nuclear chromatin related to phospholipid induced modifications of the template availability

The phospholipid involvement in the regulation of the functional and structural properties of isolated nuclei has been studied by analyzing the composition and the possible function of the nuclear matrix bound phospholipids in rat liver and murine erythroleukemic cells. The digestion of the matrix phospholipids with phospholipases results in the release of essentially all newly replicated matrix DNA. The exogenous addition of liposomal phosphatidylserine to rat liver nuclei induces chromatin structural changes consisting in a disaggregation of the heterochromatin, probably mediated by the matrix remodeling, and in a transition from the selenoid fiber to the nucleosome filament, due to the removal of the histone H1. These effects occur through a direct interaction of phospholipid molecules with the inner nuclear components, as demonstrated by carboxyfluorescein transfer and electron microscope autoradiography.