DHA Esterified to Phosphatidylserine or Phosphatidylcholine is More Efficient at Targeting the Brain than DHA Esterified to Triacylglycerol.

SCOPE: Docosahexaenoic acid (DHA, 22:6n-3) is crucial for optimal neuronal development and function, but the brain has a poor capacity to synthesize this fatty acid. When consumed acutely esterified to phosphatidylcholine, DHA is more efficient at targeting the brain than when consumed esterified to triacylglycerol. However, the brain DHA bioavailability of other forms of DHA-containing phospholipids, after oral ingestion, is unknown. The objective of this study is to compare brain uptake of DHA after acute gavage with different DHA carriers. METHODS AND RESULTS: Ten-week-old rats were gavaged with 3 H-labeled DHA esterified to phosphatidylcholine (DHA-PtdCho), phosphatidylethanolamine (DHA-PtdEtn), phosphatidylserine (DHA-PtdSer) or triacylglycerol (DHA-TG). Six hours post-gavage, the animals were euthanized and radioactivity was quantified in the cortex and serum lipid classes. Radioactivity recovered in cortex total phospholipids was similar between the DHA-PtdCho and DHA-PtdSer groups and were 5.8 and 6.7 times higher than in the DHA-TG group, respectively. Serum total lipid radioactivity was higher in the DHA-PtdSer group than in the DHA-PtdCho and DHA-PtdEtn groups, but not compared to the DHA-TG group. CONCLUSION: These results suggest that different mechanisms must be present to explain the serum and brain bioavailability differences between DHA-PtdCho and DHA-PtdSer, but these require further investigation.

Roles of PIP2 in the membrane binding of MIM I-BAR: insights from molecular dynamics simulations.

In order to probe the roles of PIP2 in the interactions between MIM I-BAR and model membranes, we performed a series of 10 µs-scale coarse-grained molecular dynamics simulations. Our results indicate that PIP2 plays predominant roles in the membrane binding of MIM I-BAR in a concentration-dependent manner and via electrostatic interactions. Besides, we find that the occurrence of the membrane curvature may induce the re-distribution of lipids in the membrane and result in the local enrichment of PIP2 at negatively curved membrane areas. Combining these roles of PIP2 in the membrane binding of MIM I-BAR helps explain how MIM I-BAR senses negative curvature and, thus, contributes to maintaining membrane protrusions.

Noninvasive in vivo multispectral optoacoustic imaging of apoptosis in triple negative breast cancer using indocyanine green conjugated phosphatidylserine monoclonal antibody.

Noninvasive and nonradioactive imaging modality to track and image apoptosis during chemotherapy of triple negative breast cancer is much needed for an effective treatment plan. Phosphatidylserine (PS) is a biomarker transiently exposed on the outer surface of the cells during apoptosis. Its externalization occurs within a few hours of an apoptotic stimulus by a chemotherapy drug and leads to presentation of millions of phospholipid molecules per apoptotic cell on the cell surface. This makes PS an abundant and accessible target for apoptosis imaging. In the current work, we show that PS monoclonal antibody tagged with indocyanine green (ICG) can help to track and image apoptosis using multispectral optoacoustic tomography

Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles.

Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Phosphatidylserine-targeted molecular imaging of tumor vasculature by magnetic resonance imaging.

Phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, becomes exposed on the outer surface of viable endothelial cells in tumor vasculature, but not in normal blood vessels. In the present study, we report the use of PGN635, a novel human monoclonal antibody that specifically targets PS, for in vivo molecular MRI of tumor vasculature. The F(ab')2 fragments of PGN635 were conjugated to polyethylene glycol (PEG) coated iron oxide nanoparticles (IO). Targeting specificity of the PS-targeted Nanoprobe, IO-PGN635F(ab')2 was first confirmed by in vitro MRI and histological staining. In vivo longitudinal MRI was then performed before and after i.v. injection of IO-PGN635F(ab')2 into mice bearing 4T1 breast tumors. T2-weighted MR images at 9.4 T revealed inhomogeneous signal loss in tumor as early as 2 h post injection. Furthermore, ionizing radiation induced a significant increase in PS exposure on tumor vascular endothelial cells, resulting in significantly enhanced and sustained tumor contrast (p < 0.05). Spatially heterogeneous MRI contrast correlated well with histological staining of tumor vascular endothelium. Our studies suggest that PS exposed within the lumen of tumor vasculature is a highly specific and useful biomarker for targeted MRI contrast agents.

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages.

BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.

ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis 1 (PFIC1) results from mutations in ATP8B1, a putative aminophospholipid flippase. Conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1 deficiency produces cholestasis by altering the activity of the farnesoid X receptor (FXR) or by impairing the structure of the canalicular membrane. METHODS: ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes and Caco2 cells using adenoviral and oligonucleotide small interfering RNAs. RESULTS: ATP8B1 messenger RNA and protein expression was greatly reduced in human and rat cells. In contrast, FXR expression and several FXR-dependent membrane transporters (bile salt export pump [BSEP], multidrug resistance-associated protein [MRP] 2) were unchanged at messenger RNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells, as evidenced by 6alpha-ethyl chenodeoxycholic acid-mediated induction of small heterodimer partner, BSEP, and multidrug-resistant protein (MDR) 3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes, although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine, consistent with the function of Atp8b1 as an aminophospholipid flippase. CONCLUSIONS: ATP8B1 deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated with cholestasis.

Fluorescent liposomes for intravital staining of Kupffer cells to aid in vivo microscopy in rats.

The potential of newly formulated fluorescent-labeled liposomes for the intravital staining of Kupffer cells was evaluated in rats. Fluorescently labeled phosphatidylcholine (PC) was incorporated into liposomes consisting of PC and phosphatidylserine. After intravenous injection, Kupffer cells in the rat liver were intravitally stained and were clearly delineated under the fluorescence image of both confocal laser scanning microscopy and in vivo microscopy. Specificity of the staining was confirmed by immunohistochemistry using the anti-rat macrophage antibody Ki-M2R, which suggested that the liposomes were selectively entrapped by the hepatic reticuloendothelial system. A time-course study revealed that the suitable observation window was between 16 and 24 h after the injection. Phagocytic activity of Kupffer cells after the administration of liposomes was examined by measuring the amount of hepatic uptake of intravenously administered fluorescent microspheres; no detrimental influence of the liposomes on the phagocytic activity was observed. Additionally, no histopathologic changes were found in the livers from liposome-treated rats. Therefore, the fluorescent-labeled liposomes appear to be a useful research tool for labeling Kupffer cells for in vivo microscopic observation of the liver.

Acute cognitive effects of standardised Ginkgo biloba extract complexed with phosphatidylserine.

Recent data suggest that the complexation of standardised Ginkgo biloba extract (GBE) with soy-derived phospholipids enhances the bioavailability of GBE's active components. The current study therefore aimed to assess the comparative cognitive and mood effects of a low dose of GBE and products complexing the same extract with either phosphatidylserine or phosphatidylcholine. The study utilised a placebo-controlled, multi-dose, double-blind, balanced-crossover design. Twenty-eight healthy young participants received 120 mg GBE, 120 mg GBE complexed with phosphatidylserine (Virtiva), 120 mg GBE complexed with phosphatidylcholine and a matching placebo, on separate days 7 days apart. Cognitive performance was assessed using the Cognitive Drug Research (CDR) computerised test battery and Serial Subtraction tasks immediately prior to dosing and at 1, 2.5, 4 and 6 h thereafter. The primary outcome measures were the four aspects of cognitive performance, which have previously been derived by factor analysis of CDR subtests. Levels of terpenoids (bilobalide, ginkgolide A and ginkgolide B) were concomitantly assessed in plasma samples taken pre-dose and at 3 and 6.5 h post-dose.In keeping with previous research utilising the same methodology, 120 mg of GBE was not associated with markedly improved performance on the primary outcomes. However, administration of GBE complexed with phosphatidylserine resulted both in improved secondary memory performance and significantly increased speed of memory task performance across all of the post-dose testing sessions. Enhancement following GBE complexed with phosphatidylcholine was restricted to a modest improvement in secondary memory performance which was restricted to one post-dose time point. All three treatments were associated with improved calmness. There were no significant differences in post-dose levels of terpenoids between the Ginkgo containing treatments, although this latter finding may be attributable to methodological factors. Complexation with phosphatidylserine appears to potentiate the cognitive effects associated with a low dose of GBE. Further research is required to identify whether this effect is due to the complexation of the extracts, their mere combination, or the separate psychopharmacological actions of the two extracts.

In vitro and in vivo characterization of a combination chemotherapy formulation consisting of vinorelbine and phosphatidylserine.

The purpose of these studies was to design an intravenous drug formulation consisting of two active agents having synergistic in vitro activity. Specifically, we describe a novel drug combination consisting of a cytotoxic agent (vinorelbine) with an apoptosis-inducing lipid (phosphatidylserine, PS). In vitro cytotoxicity screening of PS and vinorelbine, alone and in combination, against human MDA435/LCC6 breast cancer and H460 lung cancer cells was used to identify the molar ratio of these two agents required for synergistic activity. PS and vinorelbine were co-formulated in a lipid-based system at the synergistic molar ratio and the pharmacokinetic and antitumor characteristics of the combination assessed in mice bearing H460 tumors. The cytotoxicity of the lipid, and the synergy between the lipid and vinorelbine, were specific to PS; these effects were not observed using control lipids. A novel formulation of PS, incorporated as a membrane component in liposomes, and encapsulating vinorelbine using a pH gradient based loading method was developed. The PS to vinorelbine ratio in this formulation was 1/1, a ratio that produced synergistic in vitro cytotoxicity over a broad concentration range. The vinorelbine and PS dual-agent treatment significantly delayed the growth of subcutaneous human H460 xenograft tumors in Rag2M mice compared to the same dose of free vinorelbine given alone or given as a cocktail of the free vinorelbine simultaneously with empty PS-containing liposomes. These studies demonstrate the potential to develop clinically relevant drug combinations identified using in vitro drug-drug interactions combined with lipid-based delivery systems to co-formulate drugs at their synergistic ratios.

Effects of phosphatidylserine supplementation on exercising humans.

Phosphatidylserine (PtdSer) is a ubiquitous phospholipid species that is normally located within the inner leaflet of the cell membrane. PtdSer has been implicated in a myriad of membrane-related functions. As a cofactor for a variety of enzymes, PtdSer is thought to be important in cell excitability and communication. PtdSer has also been shown to regulate a variety of neuroendocrine responses that include the release of acetylcholine, dopamine and noradrenaline. Additionally, PtdSer has been extensively demonstrated to influence tissue responses to inflammation. Finally, PtdSer has the potential to act as an effective antioxidant, especially in response to iron-mediated oxidation. The majority of the available research that has investigated the effects of PtdSer supplementation on humans has concentrated on memory and cognitive function; patients experiencing some degree of cognitive decline have traditionally been the main focus of investigation. Although investigators have administered PtdSer through intravenous and oral routes, oral supplementation has wider appeal. Indeed, PtdSer is commercially available as an oral supplement intended to improve cognitive function, with recommended doses usually ranging from 100 to 500 mg/day. The main sources that have been used to derive PtdSer for supplements are bovine-cortex (BC-PtdSer) and soy (S-PtdSer); however, due to the possibility of transferring infection through the consumption of prion contaminated brain, S-PtdSer is the preferred supplement for use in humans. Although the pharmacokinetics of PtdSer have not been fully elucidated, it is likely that oral supplementation leads to small but quantifiable increases in the PtdSer content within the cell membrane.A small number of peer-reviewed full articles exist that investigate the effects of PtdSer supplementation in the exercising human. Early research indicated that oral supplementation with BC-PtdSer 800 mg/day moderated exercise-induced changes to the hypothalamo-pituitary-adrenal axis in untrained participants. Subsequently, this finding was extended to suggest that S-PtdSer 800 mg/day reduced the cortisol response to overtraining during weight training while improving feeling of well-being and decreasing perceived muscle soreness. However, equivocal findings from our laboratory might suggest that the dose required to undertake this neuroendocrine action may vary between participants.Interestingly, recent findings demonstrating that short-term supplementation with S-PtdSer 750 mg/day improved exercise capacity during high-intensity cycling and tended to increase performance during intermittent running might suggest an innovative application for this supplement. With the findings from the existing body of literature in mind, this article focuses on the potential effects of PtdSer supplementation in humans during and following exercise.

Surface charge and the association of liposomes with colon carcinoma cells.

Interaction between the plasma membrane and aggregate lipid surface determines how efficiently the encapsulated drug will be delivered into the cell. Electrostatic interactions are one of the main forces affecting liposome and aggregate association with the charged cell surface. In this study, the effect of surface charge on the association of liposomes with human colon CX-1.1 cancer cells was studied. When phosphatidylserine was incorporated into a lipid bilayer, the amount of liposomes associated with cells tended to increase along with the amount of negatively charged lipid present in the liposomal lipid bilayer. When the cationic lipid dioleoyl-1,2-diacyl-3-trimethylammonium-propane (DOTAP) was included into the liposome formula, their uptake by the cells was also increased. Maximum binding occurred when the amount of positively charged lipids in liposomes was about 10 mol% of lipids.

Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol).

Incorporation of 5 mol% poly(ethylene glycol)-conjugated lipids (PEG-lipids) has been shown to extend the circulation longevity of neutral liposomes due to steric repulsion of PEG at the membrane surface. The effects of PEG-lipids on protein interactions with biologically reactive membranes were examined using phosphatidylserine (PS) containing liposomes as the model. Incorporating 15 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 into PS liposomes resulted in circulation lifetimes comparable to that obtained with neutral liposomes containing 5 mol% DSPE-PEG 2000. These results suggested that 15 mol% DSPE-PEG 2000 may be effective in protecting PS liposomes from the high affinity, PS-mediated binding of plasma proteins. This was determined by monitoring the effects of PEG-lipids on calcium-mediated blood coagulation protein interactions with PS liposomes. Prothrombin binding and procoagulant activity of PS liposomes could be inhibited >80% when 15 mol% DSPE-PEG 2000 was used. These results are consistent with PS on membrane surfaces forming transient nucleation sites for protein binding that may result in lateral exclusion of PEG-lipids incorporated at <10 mol%. These nucleation sites may be inaccessible when PEG-lipids are present at elevated levels where they adopt a highly compressed brush conformation. This suggests that liposomes with reactive groups and PEG-lipids may be appropriately designed to impart selectivity to protein interactions with membrane surfaces.

The complement system enhances the clearance of phosphatidylserine (PS)-liposomes in rat and guinea pig.

In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig.

Oxalate-induced redistribution of phosphatidylserine in renal epithelial cells: implications for kidney stone disease.

AIMS: The present studies assessed the possibility that exposure to oxalate leads to alterations in membrane structure that promote crystal binding to renal epithelial cells. Specifically, we determined whether oxalate exposure produces a redistribution of membrane phosphatidylserine (PS) and an increase in the binding of (14)C-oxalate crystals to renal epithelial cells. METHODS: PS distribution was monitored in MDCK cells and in phospholipid-containing vesicles using NBD-PS, a fluorescent derivative of PS. Superficial PS was also detected by monitoring the binding of annexin V to MDCK cells. RESULTS: Oxalate exposure rapidly increased the abundance of superficial NBD-PS and increased the binding of annexin V to MDCK cells. Oxalate exposure also increased PS at the surface of phospholipid vesicles, suggesting that oxalate may interact directly with PS. The oxalate concentrations that increased superficial PS also increased binding of (14)C-oxalate crystals to MDCK cells, and the increased crystal binding was blocked by annexin V. CONCLUSIONS: These findings provide direct evidence that oxalate exposure promotes both a redistribution of PS and an increase in crystal binding in renal epithelial cells and support the notion that oxalate toxicity may contribute to the development of stone disease by altering the properties of the renal epithelial cell membrane.

Interactions of PC/Chol and PS/Chol liposomes with human cells in vitro.

Interactions between phosphatidylcholine (PC) or phosphatidylserine (PS) liposomes and human umbilical vein endothelial cells (HUVEC) or human promyelocytic leukemia cells (HL60) were investigated. Pyramine encapsulating or rhodamine incorporating small unilamellar liposomes with mean diameters around 80 nm (demonstrated to retain encapsulated material and to be nontoxic under experimental conditions) were used. Liposome uptake by both types of cells increased when increasing amounts of vesicles were co-incubated. For both lipid compositions, the interaction with HUVEC was very fast (association reached a plateau within 5 min) and so was the release of internalized vesicles (90% within 10 min at 37 degrees C). The reduced association values at 4 degrees C and the punctuate fluorescence observed in the cell cytoplasm after interaction, were indicative of whole liposome internalization. This internalization was clathrin-independent, since it was not inhibited by sodium azide and deoxyglucose. Pre-treatment of HUVEC with filipin or NEM resulted in modification of the interaction, something that could be due to alterations in the biochemical characteristics of HUVEC membranes that inhibit vesicular processes. In HL-60 cells, a slower association and faster release of PC/Chol liposomes was demonstrated, while association of both liposomes with these cells was energy-and temperature-independent. Nevertheless, morphological studies revealed differences in the interactions: A bright fluorescent rim observed after interaction with PC/Chol liposomes, suggests that these liposomes were adsorbed on the surface of HL60 cells, while the uniform cytoplasmic fluorescence observed after incubation with PS/Chol liposomes was indicative of fusion as the interaction mechanism.

Mercuric chloride induces apoptosis in human T lymphocytes: evidence of mitochondrial dysfunction.

The major objective of our study was to define the mechanism by which mercuric chloride (HgCl2) induces human T-cell death. Human peripheral blood T-cells were exposed to 0-40 microm HgCl2 and then analyzed for biochemical and molecular features of T-cell apoptosis. HgCl2-treated cells exhibited increased Hoechst 33258 fluorescence while maintaining their ability to exclude the vital stain 7-aminoactinomycin D. To further evaluate cell death and distinguish between apoptosis and necrosis, translocation of phosphatidylserine to the outer layer of the plasma membrane (annexin V binding), DNA fragmentation (TUNEL assay), and cleavage of poly (ADP-ribose) polymerase (PARP) were assessed. In the presence of 20-40 microm HgCl2, T-cells exhibited increased annexin V binding (28%) and DNA fragmentation (31%). HgCl2-dependent PARP cleavage was also observed by Western blot analysis. Because degradative changes associated with apoptosis are often preceded by disruption of mitochondrial function, HgCl2-treated cells were assessed for disruption of the mitochondrial transmembrane potential (DeltaPsim) and development of the mitochondrial permeability transition state. Using DiOC6(3), we demonstrated that HgCl2 exposure resulted in a decrease in the DeltaPsim. Because a decline in DeltaPsim can disturb the intracellular pH (pHi), we used the fluorescent probe, SNARF-1, to assess intracellular acidification. Treatment of T-cells with HgCl2 resulted in reduced pHi from 7.0 to 6.7. Concomitant with these observations, the fluorescent probe, hydroethidine, was utilized to demonstrate that uncoupled mitochondrial electron transport resulted in increased reactive oxygen species (ROS) generation. Interestingly, in spite of these alterations to mitochondrial function, translocation of cytochrome c to the cytosol was not detected; this correlated with enhanced bcl-2 levels in HgCl2-treated cells. In conclusion, HgCl2 exposure results in oxidative stress and activation of death signaling pathways leading to apoptosis. Collectively, our studies indicate that individual mercurial species are capable of inducing T-cell death by activating specific apoptotic cascades.

Different intrahepatic distribution of phosphatidylglycerol and phosphatidylserine liposomes in the rat.

Liposomes with diameters of 200 to 400 nm containing phosphatidylserine (PS) or phosphatidylglycerol (PG) were injected intravenously into rats. Two hours after injection, 75% of the injected dose of PS liposomes was found in the liver and only 10% found in the spleen, while 35% of the PG liposomes was found in the liver and as much as 40% was found in the spleen. Cell-isolation experiments revealed the following remarkable difference in the intrahepatic distribution between the two liposome formulations: the PS liposomes distributed in about equal amounts to Kupffer cells and hepatocytes, despite their size (200-400 nm) exceeding that of the endothelial fenestrae (average 150 nm), whereas the PG liposomes were only taken up by the Kupffer cells and not at all by the hepatocytes. Double-label studies, using liposomes in which the lipid-moiety was radio labeled with [3H]cholesteryloleylether ([3H]CE) and the water phase with [14C]sucrose, showed that the liposomes were taken up as intact particles. These observations were confirmed through electron microscopy by determining the in situ localization of liposome-encapsulated colloidal gold particles in thin sections of liver and spleen. The differences in organ distribution are ascribed to differences in opsonization patterns of the two liposomal surfaces. For the difference in intrahepatic distribution, we offer the following two explanations: the exploitation of the blood cell-mediated forced sieving concept and the indication of a PS-specific pharmacological effect on the dimensions of the fenestrations.