Cholesteryl Hexadecyl Ether Research Articles

Human variant of scavenger receptor BI (R174C) exhibits impaired cholesterol transport functions

AbstractHDL and its primary receptor, scavenger receptor class B type I (SR-BI), work together to promote the clearance of excess plasma cholesterol, thereby protecting against atherosclerosis. Human variants of SR-BI have been identified in patients with high HDL-cholesterol levels, and at least one variant has been linked to cardiovascular disease. Therefore, while often regarded as beneficial, very high levels of HDL-cholesterol may result from impaired cholesterol clearance through SR-BI and contribute to cardiovascular risk. In this study, we characterized the function of a rare human variant of SR-BI, resulting in the substitution of arginine-174 with cysteine (R174C), which was previously identified in a heterozygous individual with high levels of HDL-cholesterol. We hypothesized that the R174C-SR-BI variant has impaired cholesterol transport functions, which were assessed in COS-7 cells after transient transfection with full-length WT or R174C-SR-BI. Although R174C-SR-BI was expressed at levels comparable to the WT receptor, HDL binding, cholesteryl hexadecyl ether uptake, free cholesterol efflux, and modulation of membrane cholesterol were disrupted in the presence of R174C-SR-BI. We further examined the role of salt bridges as a potential mechanism for R174C-SR-BI dysfunction. If translatable, this human variant could lead to increased plasma HDL-cholesterol levels, impaired cholesterol clearance, and increased cardiovascular disease risk.

Blood clearance kinetics and organ delivery of medium-chain triglyceride and fish oil-containing lipid emulsions: Comparing different animal species

Medium-chain triglycerides (TG) (MCT) and fish oil (FO) TG are incorporated as the core TG component into intravenous (IV) lipid emulsions for infusion in parenteral nutrition. Bolus injections of IV emulsions, on the other hand, have emerged as a novel therapeutic approach to treat various acute disorders. However, intravascular metabolism and organ delivery of acute IV injection of emulsions containing both MCT and FO are not fully defined, nor have they been characterized across common experimental animal models. We characterized and compared blood clearance kinetics and organ distribution of bolus injections of MCT/FO emulsions among different animal species. We also examined whether sex differences or feeding status can affect catabolic properties of MCT/FO lipid emulsions. Blood clearance rates of lipid emulsions with specific TG composition were compared in rats IV injected with [3H]cholesteryl hexadecyl ether labeled pure n-6 long-chain (LCT) and n-3 FO TG lipid emulsions, or emulsions containing MCT and FO at different ratios (wt/wt), which include 8:2 (80% MCT: 20% FO), 5:4:1 (50% MCT: 40% LCT: 10% FO) and SMOF (30% LCT: 30% MCT: 25% olive oil: 10% FO). Dose-response effects (0.016mg-1.6mgTG/g body weight) of the MCT/FO 8:2 emulsions on blood clearance properties and organ delivery were determined in both mice and rats. Blood clearance kinetics and organ uptake of MCT/FO 8:2 emulsions were compared between male and female rats and between fed and fasted rats. Changes in plasma lipid profiles after acute injections of MCT/FO 8:2 lipid emulsion at different doses (0.043, 0.133, and 0.4mgTG/g body weight) were characterized in non-human primates (Cynomolgus monkeys). MCT/FO 8:2 emulsion was cleared faster in rats when compared with other emulsions with different TG contents. Mice had faster blood clearance and higher fractional catabolic rates (FCR) when compared with the rats injected with MCT/FO 8:2 emulsions regardless of the injected doses. Mice and rats had similar plasma TG and free fatty acid (FFA) levels after low- or high-dose injections of the MCT/FO emulsion. Tissue distribution of the MCT/FO 8:2 lipid emulsion are comparable between mice and rats, where liver had the highest uptake per recovered dose among all organs (>60%). Feeding status and sex differences did not alter the blood clearance rate of the MCT/FO 8:2 emulsion in rats. In a nonhuman primate model, dose-response increases in plasma TG and FFA were observed after IV injection of MCT/FO 8:2 emulsions within the 1st 10min. A lipid emulsion containing both MCT and FO TG is cleared rapidly in blood and readily available for organ uptake in rodent and primate animal models. Characterization of the blood clearance properties of the MCT/FO 8:2 emulsion administered in various animal models may provide further insight into the safety and efficacy profiles for future therapeutic use of bolus injections of MCT/FO emulsions in humans.

Radiolabeled cholesteryl ethers: A need to analyze for biological stability before use

Radiolabeled cholesteryl ethers are widely used as non-metabolizable tracers for lipoproteins and lipid emulsions in a variety of in vitro and in vivo experiments. Since cholesteryl ethers do not leave cells after uptake and are not hydrolyzed by mammalian cellular enzymes, these compounds can act as markers for cumulative cell uptakes of labeled particles. We have employed [3H]cholesteryl oleoyl ether to study the uptake and distribution of triglyceride-rich emulsion particles on animal models. However, questionable unexpected results compelled us to analyze the stability of these ethers. We tested the stability of two commercially available radiolabeled cholesteryl ethers - [3H]cholesteryl oleoyl ether and [3H]cholesteryl hexadecyl ether from different suppliers, employing in vitro, in vivo and chemical model systems. Our results show that, among the two cholesteryl ethers tested, one ether was hydrolyzed to free cholesterol in vitro, in vivo and chemically under alkaline hydrolyzing agent. Free cholesterol, unlike cholesteryl ether, can then re-enter the circulation leading to confounding results. The other ether was not hydrolyzed to free cholesterol and remained as a stable ether. Hence, radiolabeled cholesteryl ethers should be analyzed for biological stability before utilizing them for in vitro or in vivo experiments.

How Does DHA Reach the Brain to Provide Neuroprotection after Hypoxic‐Ischemic Injury?

BackgroundPrevious papers have reported that intravenously injected radiolabeled docosahexaenoic acid (DHA) rapidly enters rat brain. Recent studies from our laboratory have shown that acute intraperitoneal (i.p) injection of triglyceride (TG) emulsions rich in n‐3 fatty acids, especially tri‐DHA provides neuroprotection after hypoxic‐ischemic (HI) injury in neonatal mice (PLoS One. 2016;11(8):e0160870).AIMTo determine how injected tri‐DHA released DHA and perhaps other DHA derivatives are delivered to brain to provide neuroprotection after HI.MethodologyLaboratory made tri‐DHA emulsions were labeled with non‐metabolizable [3H]‐cholesteryl hexadecyl ether. Neonatal naïve or HI mice (C57BL/6J) were injected with radiolabeled tri‐DHA emulsion intraperitoneally (0.375 mg TG/g bw). At 4 and 24 hrs after injection, blood, peritoneal fluid, lung, liver, heart, brain, kidney, muscle, bone, spleen and adipose tissue were harvested and levels of radiolabeled emulsion in each organ determined. Tissue uptake was expressed as percent of recovered injected dose. HI was produced by right carotid artery ligation followed by hypoxia (8% O2 at 37°C) for 15 mins.ResultsNo significant differences were observed between naïve or HI mice in uptake of tri‐DHA emulsion particles at 4 hrs after injection. Among naïve and HI mice, the percent of recovered dose of radioactivity in blood and intraperitoneal fluid at 4 hrs were found to be: blood‐ 7.7 ± 5.5% naïve vs 7.4 ± 0.81% HI; intraperitoneal fluid‐ 12.6 ± 8.8% naïve vs 17.8 ± 7.6% HI. Among all organs assayed, liver contained most of the recovered dose of radiolabeled emulsion (40.5 ± 8.9% naïve vs 42.7 ± 0.1% HI). Lowest emulsion particle uptake was found to be in the brain among naïve and HI mice (<0.1% of the recovered dose among both naïve and HI groups). Compared to 4 hrs, no significant changes were observed in the uptake of emulsion particles at 24 hrs after the injection.ConclusionsThe present data demonstrates that, neonatal mice efficiently clear radiolabeled emulsion from intraperitoneal fluid after i.p. injection. Uptake of emulsion particles was high in liver and very low in brain indicating that whole emulsion particle uptake by brain does not mediate neuroprotection after HI. This suggests the involvement of other pathways in DHA mediated neuroprotection in HI brain. We also hypothesize that local neuroprotection provided by DHA in HI brain is mediated, in part through DHA derivatives produced locally in brain and in other tissues.

Scavenger receptor-BI is a receptor for lipoprotein(a)

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.

The Use of Radioactive Marker as a Tool to Evaluate the Drug Release in Plasma and Particle Biodistribution of Block Copolymer Nanoparticles

Diblock copolymer nanoparticles encapsulating a paclitaxel prodrug, Propac 7, have been used to demonstrate the usefulness of a nonmetabolizable radioactive marker, cholesteryl hexadecyl ether (CHE), to evaluate nanoparticle formulation variables. Since CHE did not exchange out of the nanoparticles, the rate of clearance of the CHE could be used as an indicator of nanoparticle stability in vivo. We simultaneously monitored prodrug circulation and carrier circulation in the plasma and the retention of CHE relative to the retention of prodrug in the plasma was used to distinguish prodrug release from nanoparticle plasma clearance. Nanoparticles labelled with CHE were also used to evaluate accumulation of nanoparticles in the tumour. This marker has provided relevant data which we have applied to optimise our nanoparticle formulations.

Development of Lipidoid–siRNA Formulations for Systemic Delivery to the Liver

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

Scavenger Receptor BI-mediated Selective Uptake Is Required for the Remodeling of High Density Lipoprotein by Endothelial Lipase

Endothelial lipase (EL) is a negative regulator of high density lipoprotein (HDL) cholesterol plasma levels, and scavenger receptor BI (SR-BI) is involved in remodeling of HDL. The present study investigates the requirement of SR-BI for the effects of EL-mediated phospholipid hydrolysis on HDL metabolism in vivo. In vitro, selective uptake from EL-modified HDL was 129% higher than selective uptake from control HDL in SR-BI-overexpressing cells (p=0.01). In vivo overexpression of human EL by means of recombinant adenovirus decreased HDL plasma levels significantly (p<0.01). Fast protein liquid chromatography analysis and agarose gel electrophoresis revealed that EL expression resulted in the generation of small pre-beta HDL particles in wild-type mice, whereas in SR-BI-/- mice small HDL were preferentially removed. In kinetic experiments the fractional catabolic rate (FCR) of HDL cholesteryl ester increased by 110% (p<0.001), and the FCR of HDL apolipoproteins increased by 64% (p<0.001) in response to EL overexpression in wild-type mice. In SR-BI-/- mice a similar increase in the HDL apolipoprotein FCR occurred (p<0.001); however, there was no further increase in HDL cholesteryl ester catabolism. The apparent whole body selective uptake was increased 3-fold by EL in wild-type mice (p<0.001), whereas there was no selective uptake in SR-BI knock-out mice. EL overexpression increased hepatic selective uptake as well as holoparticle uptake (each p<0.01) in wild-type mice, whereas in SR-BI knock-out mice only holoparticle uptake increased (p<0.01). Our results indicate that SR-BI-mediated selective uptake of HDL cholesteryl ester is essential for the remodeling of large alpha-migrating HDL particles by EL.

Inhibition of liver metastasis by all- trans retinoic acid incorporated into O/W emulsions in mice

All- trans retinoic acid (ATRA) was incorporated into lipid emulsions in an attempt to alter its distribution characteristics and improve its inhibition of liver cancer metastasis. Lipid emulsions composed of egg phosphatidylcholine, cholesterol, and soybean oil were the optimized carriers for ATRA delivery, as shown by the submicron particle size and high incorporation efficiency. The particle size and zeta potential of ATRA incorporated into emulsions were about 133 nm and −11 mV, respectively. In vitro drug release study demonstrated that the release of ATRA from emulsions was sustained in the absence and present of bovine serum albumin, suggesting that ATRA was stable when incorporated in emulsions. After intravenous administration in mice, [ 3H]cholesteryl hexadecyl ether incorporated into emulsion, which is the inherent distribution of emulsions, accumulated gradually mainly in the liver. The blood concentration and hepatic accumulation of [ 3H]ATRA incorporated into emulsion was significantly higher than that of serum dissolving [ 3H]ATRA, which represent the original distribution characteristic of free ATRA. In a murine liver metastasis model by colon adenocarcinoma, the liver metastasis number and liver weight were significantly reduced and the survival time of mice was prolonged following intravenous injection of ATRA incorporated into emulsions.

Effect of Galactose Density on Asialoglycoprotein Receptor-Mediated Uptake of Galactosylated Liposomes

Galactosylated (Gal) liposomes containing various molar ratios of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)formamide (Gal-C4-Chol) as a ligand for asialoglycoprotein receptors were prepared to study the effect of the galactose content of Gal-liposomes labeled with [3H]cholesteryl hexadecyl ether on their targeted delivery to hepatocytes. The uptake characteristics of Gal-liposomes having Gal-C4-Chol of 1.0%, 2.5%, 3.5%, 5.0%, and 7.5% were evaluated. The uptake and internalization by HepG2 cells was enhanced by the addition of Gal-C4-Chol to the Gal-liposomes. In the presence of excess galactose, the uptake of Gal-liposomes having Gal-C4-Chol of 3.5%, 5.0%, and 7.5% was inhibited suggesting asialoglycoprotein receptor mediated uptake. After intravenous injection, Gal-liposomes having Gal-C4-Chol of 3.5%, 5.0%, and 7.5%, rapidly disappeared from the blood and exhibited rapid liver accumulation with up to about 80% of the dose within 10 min whereas Gal-liposomes having low Gal-C4-Chol (1.0% and 2.5%) showed a slight improvement in liver accumulation compared with bare-liposomes. Gal-liposomes with high Gal-C4-Chol are preferentially taken up by hepatocytes and the highest uptake ratio by parenchymal cells (PC) and nonparenchymal cells (NPC) (PC/NPC ratio) was observed with Gal-liposomes having of 5.0% Gal-C4-Chol. We report here that the galactose density of Gal-liposomes prepared by Gal-C4-Chol is important for both effective recognition by asialoglycoprotein receptors and cell internalization.

Biodistribution characteristics of mannosylated and fucosylated O/W emulsions in mice

Cell-specific drug delivery is one of the most promising strategies for improving therapeutic efficiency and minimizing systemic toxicity. Carrier systems devoted to receptor-mediated targeting need to be developed. In the case of liver-non-parenchymal cell-specific targeting systems, glycosylated emulsions have been developed as carriers for lipophilic drugs and/or peptides. This present study demonstrates the in vivo disposition behaviour and pharmacokinetic characteristics of mannosylated (Man-) and fucosylated (Fuc-) emulsions incorporated with cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and its fucosylated derivatives (Fuc-C4-Chol), respectively. Man- (or Fuc-) emulsions are composed of soybean oil, EggPC and Man-C4-Chol (or Fuc-C4-Chol) in a weight ratio of 70:25:5. After intravenous administration to mice, these two types of [3H]cholesteryl hexadecyl ether (CHE)-labelled glycosylated emulsions were rapidly eliminated from the blood circulation and preferentially recovered in the liver. In contrast, bare (Bare-) emulsions composed of soybean oil:EggPC:cholesterol (Chol) in a weight ratio of 70:25:5 were more retained in the blood circulation. The hepatic uptake clearances of Man- and Fuc-emulsions were 3.3- and 4.0-times greater than that of Bare-emulsions. Interestingly, the hepatic uptake clearance of Fuc-emulsions was significantly higher that that of Man-emulsions. The uptake ratios by non-parenchymal cells (NPC) and parenchymal cells (PC) (NPC/PC ratio) for Bare-, Man- and Fuc-emulsions were found to be 0.4, 2.0 and 2.9, respectively. The hepatic uptakes of [3H]CHE-labelled Man- and Fuc-emulsions were reduced by pre-dosing with glycosylated proteins and liposomes. These results clearly support the conclusion that Man- and Fuc-emulsions are promising carrier systems for liver NPC-specific targeting via receptor-mediated mechanism.

Uptake characteristics of galactosylated emulsion by HepG2 hepatoma cells

Galactosylated (Gal) emulsions containing various molar ratios of cholesten-5-yloxy- N-(4-((1-imino-2- d-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a ligand for asialoglycoprotein receptors were prepared to study the effect of the galactose content of Gal-emulsions labeled with [ 3H]cholesteryl hexadecyl ether on their targeted delivery to hepatocytes. The uptake characteristics of Gal-emulsions having Gal-C4-Chol of 1, 3, 4, 6, and 9 mol% were evaluated in HepG2 cells which posses asialoglycoprotein receptors and NIH3T3 cells which are lack of asialoglycoprotein receptors. The uptake and internalization by HepG2 cells was enhanced by the addition of Gal-C4-Chol to the Gal-emulsions whereas the uptake of Gal-emulsions by NIH3T3 cells was not much and was comparable with that of bare-emulsions. In the presence of excess Gal-BSA, the uptake of Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% was inhibited suggesting asialoglycoprotein receptor mediated uptake. Moreover, Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% showed a slight increase in surface binding and exhibited extensive uptake and internalization into HepG2 cells. The present study strongly suggested that the Gal-emulsions are taken up by the asialoglycoprotein receptor-mediated endocytosis and galactose density of Gal-emulsions is important for effective recognition and cell internalization.

Anti-CD19-Targeted Liposomal Doxorubicin Improves the Therapeutic Efficacy in Murine B-Cell Lymphoma and Ameliorates the Toxicity of Liposomes with Varying Drug Release Rates

Some formulations of liposomal doxorubicin with intermediate rates of drug release have shown increased levels of toxicity in mice. Because antibody-mediated targeting of liposomal drugs influences the pharmacokinetics, mechanism of uptake, and selectivity of the associated drugs, we hypothesized that anti-CD19-mediated targeting of liposomal doxorubicin might moderate the toxicity of the problem formulations. Phosphatidylcholine/cholesterol liposomal formulations of doxorubicin having faster, intermediate, and slower drug release rates were prepared by altering the fatty acyl chain length or degree of saturation of the phosphatidylcholine component. Pharmacokinetic and biodistribution studies and in vivo drug release rates were determined in mice using liposomes dual labeled with [3H]cholesteryl hexadecylether and [14C]doxorubicin. Therapeutic studies were done in xenograft models of human B lymphoma (Namalwa cells). The rate of clearance of the liposomal lipid was similar for all formulations (average t1/2, 18 hours), but the rate of clearance of doxorubicin was dependent on the release rate of the formulation (t1/2, 2-315 hours). Liposomes with the slowest drug release rates showed no toxicity and exhibited therapeutic activity that was superior to the other formulations when targeted with anti-CD19; liposomes with the most rapid drug release rates also showed no toxicity but showed little therapeutic effect even when targeted. Liposomes with intermediate drug release rates exhibited varying degrees of toxicity. The toxicities could be reduced and even overcome by targeting with anti-CD19 antibodies. For these formulations, therapeutic effects were intermediate between those found for liposomes with the fastest and slowest drug release rates.

Pharmacokinetic Analysis of the Tissue Distribution of Octaarginine Modified Liposomes in Mice

We recently found that octaarginine modified liposomes (R8-Lip) can be efficiently internalized by cultured cells. The purpose of the present study was to quantitatively determine the effect of R8-density on the tissue distribution of R8-Lip in mice, using their clearance as an index. R8 was introduced in the form of stearylated R8 (STR-R8). The liposomes were composed of cholesterol and egg phosphatidylcholine and were labeled with [(3)H]cholesteryl hexadecyl ether. Various densities of R8 (3%, 10% and 30%) containing liposomes were prepared with a diameter of approximately 70-80 nm. The tissue distribution of R8-Lip was determined after their i.v. administration into mice and the effect of R8-density on tissue distribution was compared with uptake clearance, the calculated tissue distribution divided by the area under the blood concentration-time course. As results, R8-Lip were more rapidly eliminated from circulating blood and distributed to many tissues, especially liver depending on the R8-density. However, the tissue uptake clearance represented similar value to that of positively charge liposomes. Based on these results, we conclude that the R8-dependent increase in R8-Lip in various tissues tested indicates that positive charge, but not PTD function derived from R8 predominantly responsible for the enhancement of tissue distribution. Therefore, it is suggested that topology control of R8 is important to exhibit the PTD function.

Inhibition of oxidized LDL aggregation with the calcium channel blocker amlodipine: role of electrostatic interactions

Atherogenic low-density lipoproteins (LDL) are characterized by elevations in cholesterol content and increased electronegativity, factors that contribute to aggregation and foam cell formation. This study was designed to test the effect of the positively charged calcium channel blocker (CCB) amlodipine on the aggregation properties of oxidized LDL lipids. Large unilamellar vesicles (LUVs) (100 nm diameter) labeled with a non-exchangeable marker [ 3H]cholesteryl hexadecyl ether were prepared with lipids extracted from human LDL following oxidation. The LUVs were shown to bind, in a reversible fashion, to charged diethylaminoethyl Sephadex columns. The addition of amlodipine inhibited binding of the oxidized LDL lipids in a dose-dependent fashion with an IC 50 in the nanomolar range as a result of its high lipophilicity and positively charged amino group (p K a of 9.02). The activity of amlodipine was reproduced in model membranes that contained fixed amounts of charged phospholipid (glycerophospholipid) in a concentration-dependent manner. By contrast, drugs lacking a formal positive charge, including CCBs (felodipine, nifedipine, diltiazem, verapamil) and an angiotensin-converting enzyme-inhibitor (ramiprilate) had no effect on the column binding of the modified, electronegative lipids. These effects of amlodipine on LDL lipid aggregation and electrostatic properties may represent a novel antiatherosclerotic mechanism of action.

Oxidative tyrosylation of high density lipoproteins impairs cholesterol efflux from mouse J774 macrophages: role of scavenger receptors, classes A and B.

Studies were designed to test whether tyrosylation of high-density lipoprotein (HDL(T)) modifies its metabolic features. HDL(T) was less effective than native HDL in promoting cholesterol efflux from J774-AI macrophages. Cell association with fluorescent HDL(T)-apolipoprotein and the uptake of HDL(T)-[(3)H]cholesteryl hexadecyl ether were enhanced by 50% in comparison with native HDL. In addition, neutral cholesterol ester hydrolase (nCEH) activity in J774-AI, which controls the hydrolysis of cholesteryl ester stores to provide free cholesterol for cellular release, declined in the presence of HDL(T). In vitro displacement experiments revealed the ability of HDL(T) to compete with oxidized and acetylated LDL, known as ligands of scavenger receptor (SR) class B type I/II. Similarly, treatment with a blocking antibody to SR-BI/II reduced the cell association of HDL(T) and native HDL by 50%. The addition of polyinosinic acid, an inhibitor of SR class A, reduced the cell association of HDL(T) without affecting that of native HDL. These findings provide evidence that HDL(T) can compete with modified LDL, bind SR-BI/BII and internalize cholesterol ester. Furthermore, the impaired capacity of HDL(T) in promoting cholesterol efflux from J774-AI was accompanied by diminished nCEH and enhanced recognition by SR-AI/II, which appears to involve the transport of cholesterol into cells.

Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes

To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5–10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG 2000 (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG 2000 (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using ‘empty’ and drug-loaded [ 3H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG 2000 or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC 0–24h) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC 0–4h for free IDA was 0.030 μmole h/ml as compared to 1.38 μmole h/ml determined for the DSPC:DSPE-PEG 2000 formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes.

Inhibition of liver metastasis by targeting of immunomodulators using mannosylated liposome carriers

Mannosylated liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-β-d-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar liposomes consisting of cholesterol and distearoyl phosphatidylcholine (DSPC). The biodistribution of liposomes labeled with [3H]cholesteryl hexadecyl ether was examined in mice. The rate and extent of the hepatic uptake of those [3H]liposomes increased proportionally on increasing the mixing ratio of Man-C4-Chol. Their hepatic uptake was reduced by increasing the administered dose due to the limited number of mannose receptors. The liver uptake of [3H]Man-liposomes was preferentially mediated by liver non-parenchymal cells (NPC) and significantly inhibited by co-injection with an excess of Man-BSA, indicating the involvement of a mannose receptor-mediated mechanism in the hepatic uptake of Man-liposomes. Muramyl dipeptide (MDP), an immunomodulator, was also incorporated into the liposomes and its inhibitory effect in an experimental liver metastasis model was examined. In contrast to free MDP treatment, which showed little effect on the inhibition of metastasis, liposomal MDP significantly reduced the number of metastatic colonies in the liver. Active targeting of MDP to liver NPC by Man-liposomes resulted in more effective inhibition than delivery of MDP by liposomes without mannose. Treatment with MDP/Man-liposomes further increased the survival of the tumor-bearing mice. These results suggest that Man-liposomes are effective carriers for targeted delivery of bioactive compounds to liver NPC.

Novel Galactosylated Liposomes for Hepatocyte‐Selective Targeting of Lipophilic Drugs

Novel galactosylated neutral liposomes containing cholesten‐5‐yloxy‐N‐(4‐((1‐imino‐2‐β‐D‐thiogalactosylethyl)amino)butyl)formamide (Gal‐C4‐Chol) as a “homing” device were developed for hepatocyte‐selective drug targeting. Distearoylphosphatidylcholine (DSPC)/cholesterol (Chol) (60:40) and DSPC/Chol/Gal‐C4‐Chol (60:35:5) liposomes were prepared and labeled with [3H]cholesteryl hexadecyl ether (CHE). [3H]Prostaglandin E1 (PGE1) and [14C]probucol were incorporated in liposomes as model lipophilic drugs. After intravenous injection of the liposomes, mice were sacrificed at suitable time periods, and the lung, liver, kidney, spleen, and heart were excised. DSPC/Chol/Gal‐C4‐Chol liposomes rapidly disappeared from the blood, and 85% of the dose had accumulated in the liver within 10 min compared with hepatic accumulation of DSPC/Chol liposomes of 12%. The liver was perfused with collagenase, and liver parenchymal cells (PC) and liver nonparenchymal cells (NPC) were separated by centrifugal differentiation to determine the cellular distribution. The PC/NPC ratios for DSPC/Chol/Gal‐C4‐Chol and DSPC/Chol liposomes were 15.1 and 1.1, respectively. The hepatic uptake of DSPC/Chol/Gal‐C4‐Chol liposomes, but not that of DSPC/Chol liposomes, was significantly inhibited by the predosing of galactosylated bovine serum albumin. [14C]Probucol and [3H]PGE1 incorporated in DSPC/Chol/Gal‐C4‐Chol liposomes was also efficiently delivered to the liver. In conclusion, newly developed galactosylated liposomes have been proven to be a useful carrier for hepatocyte‐selective targeting that will have many practical applications. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:105–113, 2001

Controlled biodistribution of galactosylated liposomes and incorporated probucol in hepatocyte-selective drug targeting

Two types of galactosylated liposomes containing cholesten-5-yloxy- N-(4-((1-imino-2-β- d-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a homing device were prepared to study the biodistribution of liposomal carriers and the incorporated drug. Distearoylphosphatidylcholine (DSPC)/cholesterol (Chol)/Gal-C4-Chol (60:35:5) (Gal DSPC), DSPC/Chol (60:40) (DSPC), egg yolk phosphatidylcholine (eggPC)/Chol/Gal-C4-Chol (60:35:5) (Gal eggPC), and eggPC/Chol (60:40) (eggPC) liposomes labeled with [ 3H]cholesteryl hexadecyl ether (CHE) were tested and [ 14C]probucol, with a partition coefficient between octanol and water (PC oct) of 10 10.8, was selected as a model drug with lipophilicity suitable for liposomal incorporation. After intravenous injection of the combination of [ 14C]probucol and [ 3H]liposomes, the liver uptake of [ 3H]CHE was the highest in Gal DSPC liposomes, followed by Gal egg PC liposomes, egg PC liposomes, and DSPC liposomes in that order. [ 14C]Probucol incorporated in Gal DSPC liposomes exhibited lower liver uptake than [ 3H]CHE, suggesting that substantial release from liposomes had taken place. In contrast, [ 14C]probucol incorporated in Gal eggPC liposomes was more stably incorporated under in vivo conditions. Co-administration with galactosylated bovine serum albumin significantly inhibited the liver uptake of [ 14C]probucol in both types of galactosylated liposomes, suggesting that the hepatic uptake of liposomes should be mediated by asialoglycoprotein receptors being [ 14C]probucol incorporated in them.