Plasma lipidomic signatures of spontaneous obese rhesus monkeys.

BACKGROUND: Obesity plays crucial roles in the pathogenesis of metabolic diseases such as hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), and type 2 diabetes (T2D). The underlying mechanisms linking obesity to metabolic diseases are still less understandable. METHODS: Previously, we screened a group of spontaneously obese rhesus monkeys. Here, we performed a plasma lipidomic analysis of normal and obese monkeys using gas chromatography/mass spectroscopy (GC/MS) and ultra-high performance liquid chromatography/mass spectroscopy (UPLC/MS). RESULTS: In total, 143 lipid species were identified, quantified, and classified into free fatty acids (FFA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), and sphingomyelin (SM). Data analysis showed that the obese monkeys had increased levels of fatty acids palmitoleic acid (C16:1) and arachidonic acid (C20:4), FFA especially palmitic acid (C16:0), as well as certain PC species and SM species. Surprisingly, the plasma level of LPA-C16:0 was approximately four-fold greater in the obese monkeys. Conversely, the levels of most PE species were obviously reduced in the obese monkeys. CONCLUSION: Collectively, our work suggests that lipids such as FFA C16:0 and 16:0-LPA may be potential candidates for the diagnosis and study of obesity-related diseases.

False Prolongation of Prothrombin Time in the Presence of a High Blood Concentration of Daptomycin.

Prothrombin time (PT) can reportedly be falsely prolonged by the antimicrobial drug daptomycin (DAP), and concomitant use of phosphatidylglycerol (PG). Although high doses of DAP (>6 mg/kg/day) are recommended for severe infection and result in a high blood concentration, the extent to which high blood concentrations of DAP interfere with PT, in the presence or absence of PG, has yet to be determined when using the HemosIL RecombiPlasTin 2G (Werfen Japan, Tokyo, Japan). We examined the effects of high doses of DAP on PT using this reagent. DAP (0-500 mg/L) was added to normal plasma and plasma with an already prolonged PT in the presence or absence of liposomal amphotericin B (L-AMB, 5-50 mg/L) or COATSOME EL-01 empty cationic liposomes (CS, 25-250 mg/L). Furthermore, we undertook a Monte Carlo simulation to calculate the probability of achieving DAP concentrations >100, >200 and >500 mg/L 0-48 hr after administering 6-12 mg/kg of DAP. Apparent PT increased with increasing DAP concentration, but neither L-AMB nor CS appeared to further elevate PT when co-administered with DAP. The probability of achieving DAP concentrations >100 and >200 mg/L increased with DAP dose. Higher doses of DAP than the approved dose caused false prolongation of PT. PT should be monitored carefully in patients taking high doses of DAP; ideally, PT should be measured at the trough blood concentration of DAP. Concomitant use of L-AMB and CS did not generally further elevate PT when co-administered with DAP.

Comprehensive lipid profiling of plasma in patients with benign breast tumor and breast cancer reveals novel biomarkers.

Abnormal lipid metabolism is a common feature in most solid tumors, and occurs in early stages of the tumor progression. As benign breast tumor is different from malignant tumor of breast cancer, it is particularly important to take benign breast tumor into consideration when investigating cancer biomarkers. In this study, by using a normal-phase/reversed-phase two-dimensional liquid chromatography-mass spectrometry (NP/RP 2D LC-MS) method, we conducted comprehensive lipid profiling in human plasma obtained from six benign breast tumor patients and five breast cancer patients, as well as nine healthy controls. As a result, 512 lipid species were successfully identified. Principal component analysis allowed clear separation of the three groups. Quantitative analysis revealed that many lipid contents were similar in benign and malignant breast tumors compared with controls, and these were proposed as potential breast tumor biomarkers other than breast cancer biomarkers. Two phosphatidylinositol (PI) species, including PI (16:0/16:1) and PI (18:0/20:4), could differentiate between benign and malignant breast tumors, as well as breast cancer patients and healthy controls, indicating that they could be utilized as potential breast cancer biomarkers. In addition, PI (16:0/18:1), phosphatidylglycerol (36:3), and glucosylceramide (d18:1/15:1) were demonstrated to be potential biomarkers to evaluate the level of malignancy of breast tumor. Taken together, our results indicate the usefulness of lipid profiling in the discrimination between patients with breast cancer and non-carcinoma lesions, which might provide assistance in clinical diagnosis.

Comparative plasma lipidome between human and cynomolgus monkey: are plasma polar lipids good biomarkers for diabetic monkeys?

BACKGROUND: Non-human primates (NHP) are now being considered as models for investigating human metabolic diseases including diabetes. Analyses of cholesterol and triglycerides in plasma derived from NHPs can easily be achieved using methods employed in humans. Information pertaining to other lipid species in monkey plasma, however, is lacking and requires comprehensive experimental analysis. METHODOLOGIES/PRINCIPAL FINDINGS: We examined the plasma lipidome from 16 cynomolgus monkey, Macaca fascicularis, using liquid chromatography coupled with mass spectrometry (LC/MS). We established novel analytical approaches, which are based on a simple gradient elution, to quantify polar lipids in plasma including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucocyl-ceramide, GluCer; ganglioside mannoside 3, GM3). Lipidomic analysis had revealed that the plasma of human and cynomolgus monkey were of similar compositions, with PC, SM, PE, LPC and PI constituting the major polar lipid species present. Human plasma contained significantly higher levels of plasmalogen PE species (p<0.005) and plasmalogen PC species (p<0.0005), while cynomolgus monkey had higher levels of polyunsaturated fatty acyls (PUFA) in PC, PE, PS and PI. Notably, cynomolgus monkey had significantly lower levels of glycosphingolipids, including GluCer (p<0.0005) and GM(3) (p<0.0005), but higher level of Cer (p<0.0005) in plasma than human. We next investigated the biochemical alterations in blood lipids of 8 naturally occurring diabetic cynomolgus monkeys when compared with 8 healthy controls. CONCLUSIONS: For the first time, we demonstrated that the plasma of human and cynomolgus monkey were of similar compositions, but contained different mol distribution of individual molecular species. Diabetic monkeys exhibited decreased levels of sphingolipids, which are microdomain-associated lipids and are thought to be associated with insulin sensitivity. Significant increases in PG species, which are precursors for cardiolipin biosynthesis in mitochondria, were found in fasted diabetic monkeys (n = 8).

Mass spectrometric analysis of lipid species of human circulating blood cells.

Circulating blood cell lipid composition may become increasingly important to provide new insights into cellular lipid abnormalities in diseases. Here we compared lipid species in monocytes, lymphocytes, granulocytes, platelets and red blood cells (RBC) of healthy volunteers using electrospray ionization tandem mass spectrometry and detected striking differences among the examined blood cells. The different cell types were characterized by unique lipid class and lipid species pattern. The predominant lipid classes were phosphatidylcholine (PC) and free cholesterol (FC) with cell type specific PC/FC ratios as markers of membrane fluidity which was 1.9 in monocytes, 1.3 in lymphocytes, 1.1 in granulocytes, 0.8 in platelets and 0.3 in RBC, respectively. Beside a three-fold elevated ceramide level of 2.6 mol%, granulocytes revealed the highest percentage of phosphatidylethanolamine-based plasmalogens and a decreased fraction of highly polyunsaturated (> or =3 double bonds) species compared to other cell types. Furthermore RBC showed a remarkable shift of glycerophospholipid chain length and platelets a nearly 4-fold increase of the cholesterol ester (CE) 18:2 (linoleic acid) fraction (55 mol% of total CE). In conclusion, the current study is a detailed comparison of lipid species in circulating blood cells of healthy human donors. This work could be a reference for studies in different patient cohorts directed towards discovery of novel lipid biomarkers in circulating blood cells.

Penetration of amphotericin B lipid formulations into pleural effusion.

The penetration of the amphotericin B (AMB) lipid formulations (liposomal AMB, AMB colloidal dispersion, and AMB lipid complex formulations) into pleural effusions in seven critically ill patients was assessed. AMB was detected in all pleural effusion samples at concentrations ranging from 0.02 to 0.43 microg/ml. The penetration ratio was 3 to 44%.

Role of phospholipid transfer protein on the plasma distribution of amphotericin B following the incubation of different amphotericin B formulations.

PURPOSE: The purpose of this study was to investigate the role of phospholipid transfer protein (PLTP) on the plasma distribution of amphotericin B (AmpB) following incubation with different AmpB formulations in human plasmas with varying lipid profiles. METHODS: In a first set of experiments, plasma distribution profiles of AmpB were determined following the incubation of Fungizone and lipid-based formulations (Abelcet and AmBisome) at a concentration of 20 microg AmpB/mL for 5-120 min at 37 degrees C in the plasma obtained from six different individuals (total cholesterol concentrations range between 62 and 332 mg/dL). In a second set of experiments, Abelcet, and AmBisome at a concentration of 20 microg AmpB/mL were incubated for 5 min at 37 degrees C in human plasma (total cholesterol = 163 mg/dL) that had been pretreated with an antibody raised up against PLTP (1:400 v/v dilution from stock solution) for 20 min at 37 degrees C. Following incubation, the human plasma was separated into its lipoprotein and lipoprotein-deficient fractions by density gradient ultracentrifugation and analyzed for AmpB content by high-performance liquid chromatography. RESULTS: The majority of AmpB was covered in the lipoprotein-deficient plasma and high-density lipoprotein (HDL) fractions following incubation of Fungizone in human plasma. The majority of AmpB (48.7-87.2%) was recovered in the HDL fraction following incubation of Abelcet and AmBisome in human plasma. The presence of the PLTP antibody resulted in a 20% decrease in the percentage AmpB recovered in the HDL fraction following the incubation of Abelcet. However, the plasma distribution of AmpB remained unchanged following the incubation of AmBisome in plasma containing the PLTP antibody. CONCLUSIONS: Taken together, these findings suggest indirect evidence that PLTP may play an important role in the plasma distribution profile of AmpB following the incubation of Abelcet and may be one of the factors responsible for the preferential association of AmpB with HDL when administered as Abelcet.

Population pharmacokinetics of amphotericin B lipid complex in neonates.

The pharmacokinetics of amphotericin B lipid complex (ABLC) were investigated in neonates with invasive candidiasis enrolled in a phase II multicenter trial. Sparse blood (153 samples; 1 to 9 per patient, 1 to 254 h after the dose) and random urine and cerebrospinal fluid (CSF) samples of 28 neonates (median weight [WT], 1.06 kg; range, 0.48 to 4.9 kg; median gestational age, 27 weeks; range, 24 to 41 weeks) were analyzed. Patients received intravenous ABLC at 2.5 (n = 15) or 5 (n = 13) mg/kg of body weight once a day over 1 or 2 h, respectively, for a median of 21 days (range, 4 to 47 days). Concentrations of amphotericin B were quantified as total drug by high-performance liquid chromatography. Blood data for time after dose (TAD) of <24 h fitted best to a one-compartment model with an additive-error model for residual variability, WT0.75 (where 0.75 is an exponent) as a covariate of clearance (CL), and WT as a covariate of volume of distribution (V). Prior amphotericin B, postnatal age, and gestational age did not further improve the model. The final model equations were CL (liters/h) = 0.399 x WT(0.75) (interindividual variability, 35%) and V (liters) = 10.5 x WT (interindividual variability, 43%). Noncompartmental analysis of pooled data with a TAD of >24 h revealed a terminal half-life of 395 h. Mean concentrations in the urine after 1, 2, and 3 weeks ranged from 0.082 to 0.430 microg/ml, and those in CSF ranged from undetectable to 0.074 microg/ml. The disposition of ABLC in neonates was similar to that observed in other age groups: weight was the only factor that influenced clearance. Based on these results and previously published safety and efficacy data, we recommend a daily dosage between 2.5 and 5.0 mg/kg for treatment of invasive Candida infections in neonates.

Amphotericin B lipid formulations in critically ill patients on continuous veno-venous haemofiltration.

OBJECTIVES: The pharmacokinetics of lipid-formulated amphotericin B (AMB), and of AMB that has dissociated from its lipid moiety and bound to lipoproteins in plasma, were separately determined in critically ill patients. PATIENTS AND METHODS: Eleven patients required continuous veno-venous haemofiltration (CVVH). Five of them were treated with liposomal AMB (AmBisome) and seven with AMB colloidal dispersion (Amphocil). Six of the critically ill were not undergoing CVVH (three of them treated with liposomal AMB and three with AMB colloidal dispersion). RESULTS: Significant amounts of AMB are liberated from liposomes or colloidal dispersion during circulation in plasma, where pharmacokinetics mimic that of AMB deoxycholate. Elimination of the remaining lipid-formulated fraction is different and differentially affected by CVVH. Plasma levels of lipid-formulated AMB were significantly higher in patients treated with liposomal AMB than in those treated with AMB colloidal dispersion; clearance of liposomal AMB is enhanced by haemofiltration, whereas elimination of AMB colloidal dispersion is not significantly affected. CONCLUSIONS: The pharmacokinetics of AMB that has been liberated from its lipid moiety is similar under treatment with either liposomal AMB or AMB colloidal dispersion. Since no significant influence of haemofiltration on the pharmacokinetics of liberated AMB has been found, a standard dose of lipid-formulated AMB can be recommended for patients on haemofiltration.

Preferential distribution of amphotericin B lipid complex into human HDL3 is a consequence of high density lipoprotein coat lipid content.

The purpose of this study was to determine the plasma lipoprotein (LP) distribution of amphotericin B (AmpB) and amphotericin B lipid complex [ABLC; Abelcet composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG)] and define the relationship between LP lipid concentration and composition and the distribution of AmpB and ABLC in human plasma with varying total and lipoprotein cholesterol and triglycerides. AmpB and ABLC at a concentration of 20 microg amphotericin B/mL were incubated in plasma obtained from different human subjects (n = 7) for 60 min at 37 degrees C. Following these incubations plasma samples were separated into their high-density lipoprotein (HDL), triglyceride-rich lipoprotein (TRL; which contains very low-density lipoproteins and chylomicrons), low-density lipoprotein (LDL), and lipoprotein-deficient (LPDP) fractions by density-gradient ultracentrifugation (UC) and each fraction was assayed for AmpB using high-pressure liquid chromatography (HPLC). The HDL fraction was further separated into its HDL3 and HDL2 subclasses by UC and assayed for AmpB using HPLC. Separation of HDL into its subclasses was confirmed by gel electrophoresis. To assess the influence of modified lipoprotein concentrations and lipid composition on the plasma distribution of AmpB and ABLC, these compounds were incubated in plasmas from human subjects with varying total and lipoprotein lipid concentrations. In addition, to demonstrate that alterations in HDL lipid composition influence the plasma distribution of ABLC, ABLC (20 microg amphotericin B/mL) was incubated in plasma pretreated with dithionitrobenzoate (DTNB, a compound which inhibits lecithin:cholesterol acyltransferase conversion of HDL3 free cholesterol to esterified cholesterol) 18 h prior to the experiment or in untreated plasma for 60 min at 37 degrees C. Total plasma and lipoprotein cholesterol (TC), free cholesterol (fC), esterified cholesterol (CE), triglyceride (TG), phospholipid (PL), and protein (TP) concentrations in each human sample were determined by enzymatic assays. When AmpB was incubated in human plasmas of varying lipid concentrations, the majority of the drug was recovered in the LPDP fraction. However, the majority of AmpB was recovered in the HDL3 fraction following the incubation of ABLC. Differences in lipid coat content (fC and PL) carried by HDL influenced the distribution of ABLC within plasma of different human subjects. These findings were confirmed by the DTNB treatment experiments. These findings suggest that the association of AmpB with DMPC and DMPG to form drug-lipid complexes modifies the plasma distribution of the AmpB. In addition, the distribution of ABLC among plasma lipoproteins of different human subjects is defined by the HDL lipid coat content and is possibly an important consideration when evaluating the pharmacokinetics, toxicity, and activity of these compounds following administration to humans with differing plasma lipid concentrations.

Polyethylene glycol-modified liposome-encapsulated hemoglobin: a long circulating red cell substitute.

A major obstacle in the development of red cell substitutes has been overcoming their short circulation persistence. In this study, distearoyl phosphoethanolamine polyethylene glycol 5000 (PEG-PE) (10 mol%) was added to the formulation of liposome-encapsulated hemoglobin (LEH) to decrease reticuloendothelial system uptake and prolong LEH circulation persistence. PEG-LEH was radiolabeled with technetium-99m, infused into rabbits (25% of blood pool at 1 ml/min) (n = 5), and monitored by scintigraphic imaging at various times out to 48 h. At 48 h, animals were sacrificed, and tissue samples were collected for counting in a scintillation well counter. Tissue distribution data at 48 h revealed that 51.3 +/- 3.4% of the technetium-99m-PEG-LEH remained in circulation, a greater than 3-fold increase in the circulation half-life compared with circulation half-lives previously reported for non-PEG-containing LEH formulations. The liver had the greatest accumulation at 48 h (12.7 +/- 0.7%), followed by bone marrow (6.2 +/- 0.1%), whereas the spleen had only 1.4 +/- 0.2%. The addition of PEG-PE to the LEH formulation greatly prolongs the circulation persistence of LEH and represents a significant step in the development of red cell substitutes with prolonged oxygen delivery.

Pharmacokinetic profile of ABELCET (amphotericin B lipid complex injection): combined experience from phase I and phase II studies.

Amphotericin B (AmB) has been the most effective systemic antifungal agent, but its use is limited by the dose-limiting toxicity of the conventional micellar dispersion formulation (Fungizone). New formulations with better and improved safety profiles are being developed and include ABELCET (formerly ABLC), but their dispositions have not been well characterized; hence, the reason for their improved profiles remains unclear. This report details the pharmacokinetics of ABELCET examined in various pharmacokinetic and efficacy studies by using whole-blood measurements of AmB concentration performed by high-pressure liquid chromatography. The data indicated that the disposition of AmB after administration of ABELCET is different from that after administration of Fungizone, with a faster clearance and a larger volume of distribution. It exhibits complex and nonlinear pharmacokinetics with wide interindividual variability, extensive distribution, and low clearance. The pharmacokinetics were unusual. Clearance and volume of distribution were increased with dose, peak and trough concentrations after multiple dosings increased less than proportionately with dose, steady state appeared to have been attained in 2 to 3 days, despite an estimated half-life of up to 5 days, and there was no evidence of significant accumulation in the blood. The data are internally consistent, even though they were gathered under different conditions and circumstances. The pharmacokinetics of ABELCET suggest that lower concentrations in blood due to higher clearance and greater distribution may be responsible for its improved toxicity profile compared to those of conventional formulations.

Behavior of amphotericin B lipid complex in plasma in vitro and in the circulation of rats.

Amphotericin B lipid complex (ABLC) shows reduced toxicity relative to that of amphotericin B deoxycholate (AmB-d) while maintaining antifungal activity. Rat blood or plasma was spiked with ABLC in vitro. Released amphotericin B was separated from the parent material by centrifugation. At early times (0 to 15 min) most (approximately 90%) of the amphotericin B was complexed. The amount of released amphotericin B increased gradually in a time- and temperature-dependent fashion. The released amphotericin B was associated with plasma lipoprotein and nonlipoprotein proteins. The area under the concentration-time curve from 0 to 24 h for total amphotericin B in whole blood of rats given a single intravenous bolus dose of 1 mg of ABLC per kg of body weight was fourfold lower than that in rats given 1 mg of AmB-d per kg. The complexed amphotericin B was rapidly removed from the circulation and was distributed to the tissues in these rats. Other rats were treated intravenously with ABLC (10 mg/kg/day) or AmB-d (0.5 mg/kg/day) daily for 15 days. Blood was collected at 15 and 180 min after administration of the last dose. The total levels of amphotericin B in the blood of the group given ABLC were about three to five times those in the group given AmB-d, and the concentration of released, protein-bound amphotericin B in the plasma of the group given ABLC was about one to two times that observed for the group given AmB-d, despite the 20-fold difference in dose. The relative protein distribution of amphotericin B in plasma was similar after ABLC or AmB-d administration under these steady-state conditions in vivo. The rapid uptake of complexed amphotericin B by tissues and the very low levels of circulating protein-bound amphotericin B in plasma after the administration of ABLC may explain, in part, the reduced toxicity and enhanced therapeutic index of this preparation.

Improved separation and determination of phospholipids in animal tissues employing solid phase extraction.

Separation of five glycerophospholipids having different polar groups, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) and cardiolipin (CL), was investigated by means of solid-phase extraction (SPE) cartridges. First, the phospholipids were retained in an aminopropyl-bonded phase (NH2) cartridge and subsequently eluted as neutral (PC and PE) and acidic (PS, PG and CL) glycerophospholipid fractions. Secondly, a combination of silica gel (SI) cartridge and NH2 cartridge was employed to separate five glycerophospholipids. The polarity of the eluent was responsible for neutral glycerophospholipid separation. Concerning acidic glycerophospholipids, the separation of PG and CL from PS depended mainly on the pH of the eluents, and the separation of PG and CL was affected by the solvent, depending on eluent polarities. Favorable recovery (not less than 95%, for five authentic phospholipids, 10-100 micrograms each) and repeatability (sigma = 2.3 for 10 micrograms ranges) were attained by the present method. This method of separation was applicable to the analysis of phospholipids in biological samples.

NMR lipid profiles of cells, tissues, and body fluids: proton NMR analysis of human erythrocyte lipids.

One- and two-dimensional high resolution NMR spectroscopy was applied to determine quantitatively and qualitatively the lipids extracted from human erythrocyte membranes. The relative amounts of the major lipids were determined from the spectra of unfractionated lipid extracts. After HPLC fractionation of the lipid extracts and NMR analysis of the fractions, it was possible to determine the features of the component lipids of each lipid class and to compare, especially, the fatty acid types and composition of the individual major glycerophospholipids. The results of this proton NMR analysis were compared to those obtained elsewhere using classical lipid analytical techniques and found to be in substantial agreement.

Roles of liposome composition and temperature in distribution of amphotericin B in serum lipoproteins.

The role of liposome composition and temperature in the distribution of amphotericin B (AmB) with serum lipoproteins and the role of particle charge in AmB transfer to serum lipoproteins were determined. Serum obtained from healthy volunteers was incubated with known concentrations of AmB or different liposomal formulations of AmB (1 to 100 micrograms/ml) at 37 degrees C for various time intervals (5, 10, 20, 30, 45, and 60 min). After each interval, serum was removed and separated into high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions by an LDL-direct assay. The distribution of AmB (Fungizone) at 5 min through 1 h of incubation at 25 degrees C remained constant and was similar in the HDL and LDL fractions. At 37 degrees C, at 5 through 45 min of incubation, 54 to 61% of AmB was recovered in the HDL fraction; however, at 1 h more than 75% of the AmB concentration was recovered in the HDL fraction. In contrast, 87.5 to 92% AmB was recovered in the HDL fraction throughout the incubation when negatively charged liposomal AmB (dimyristoylphosphatidylcholine [DMPC]:dimyristoylphosphatidylglycerol [DMPG], 7:3 [wt/wt]) was used. With positively charged liposomes, 75 to 87.7% of AmB was recovered in the HDL fraction through the different time points studied. AmB incorporated into DMPC (neutral) and DMPG (negative) liposomes, and AmB was distributed in an HDL:LDL ratio of 6:4 following 1 h of incubation. Ninety percent of AmB and 80% of the lipid were found in the HDL fraction in a 3:1 molar DMPG:AmB ratio and in the LDL fraction in a 6:1 molar ratio. Lipid charge and temperature play a role in AmB distribution into serum lipoproteins. AmB and DMPG may contransfer as an intact drug-lipid complex to serum lipoproteins.

Liposomal modification with uronate, which endows liposomes with long circulation in vivo, reduces the uptake of liposomes by J774 cells in vitro.

For overcoming rapid removal of liposomes from the bloodstream, we developed reticuloendothelial system (RES)-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA). In this current study, we examined the in vitro interaction of PGlcUA-liposomes with J774 cells derived from mouse macrophages. Liposomal association with J774 cells at 37 degrees C did not increase compared with the binding at 4 degrees C when liposomes were modified with PGlcUA. RES-avoiding ability was not specifically endowed by glucuronate but by uronates in general, since palmityl-D-galacturonide showed a similar effect on liposomal clearance in vivo and liposomal uptake in vitro. These facts indicate that modification of the liposomal surface with uronic acid derivatives endows liposomes with a long circulation time in the bloodstream by reducing their uptake by macrophages.

The binding of phosphatidylglycerol liposomes to rat platelets is mediated by complement.

Previous work has shown that intravenous administration of phosphatidylglycercol (PG) containing liposomes to rats results in a rapid transient decline in platelet count (1). Here the interactions of PG liposomes with rat platelets in vitro have been examined with the aim of characterizing factors associated with the decline. It is shown that PG liposomes induce formation of rat (but not human) platelet-liposome microaggregates in vitro. The PG liposome dependent thrombocytopenia observed in vivo can therefore be attributed to sequestration of PG liposome-platelet aggregates. Further, the aggregation of platelets with PG liposomes, which can be monitored as a reduction in platelet count using a coulter counter, is shown to be mediated by a serum complement factor, likely C3b. This is indicated by a requirement of plasma for the in vitro reduction in platelet count induced by PG liposomes, and the inhibition of this effect by heat treatment of plasma, by incubation of plasma with purified cobra venom factor, or by removal of C3 from plasma.

Characterization of temperature-dependent drug release from liposomes using electron spin resonance.

Liposome blood clearance and temperature-dependent drug release were studied using an electron spin resonance technique on whole blood samples. Temperature-sensitive liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1 by weight) were formed by reversed-phase evaporation. The liposomes were stable in the blood for greater than 2 h after iv injection. Liposome blood clearance exhibited a fast and slow component which may be due to the presence of both large and small liposomes in the preparation. The onset temperature for liposome release of the water-soluble nitroxide spin label was reduced from 40 degrees C in Hepes buffered saline to 38 degrees C in the rat blood, and the total release in the blood was approximately 25% greater than that measured in saline. These results show the influence of blood constituents on temperature-dependent drug release from liposomes.