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The in vivo association of blood proteins with large unilamellar liposomes composed of saturated phosphatidylcholines was analyzed to determine the effect of membrane fluidity and hydrocarbon chain length on liposome-plasma protein interactions and liposome clearance. Liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diarachidoylphosphatidylcholine (DAPC) were administered via the lateral tail vein of CD-1 mice and were subsequently isolated from the blood at 2 min postinjection. The protein binding ability (PB, grams of protein bound per mole total lipid) of the liposomes was quantified and related to their circulation half-lives. Liposomes composed of long-chain saturated phospholipids that exist in the gel (frozen) state at 39 degrees C (DPPC,DSPC and DAPC) bound large quantities of blood proteins, in excess of 48 g of protein per mole total lipid, and were found to be rapidly cleared from the circulation. The incorporation of cholesterol into DSPC liposomes resulted in significantly decreased PB values and enhanced circulation lifetimes for this lipid system. This cholesterol effect plateaued at 30 mol % cholesterol, corresponding to the loss of the gel-liquid crystalline phase transition, and resulted in PB values of 23-28 grams of protein per mole of total lipid. The types of blood proteins binding to DSPC liposomes were not significantly altered by the inclusion of cholesterol. This is the first demonstration of rapid clearance of neutral large unilamellar liposomes having high levels of bound protein.

The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20 fold higher than that of a CMV nuclear expression plasmid control. Here we describe various strategies to increase the levels of autogene based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES sequence. It was found that none of these changes demonstrated a significant increase in gene expression. However determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments, revealed transgene mRNA levels up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production is a major factor limiting autogene based cytoplasmic expression.

Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e139; doi:10.1038/mtna.2013.66; published online 17 December 2013.

Liposomes recovered from the blood of liposome-treated CD1 mice were previously reported to have a complex protein profile associated with their membranes (Chonn, A., Semple, S.C., and Cullis, P.R. (1992) J. Biol. Chem. 267, 18759-18765). In this study, we have further characterized and identified the major proteins associated with very rapidly cleared large unilamellar vesicles. These liposomes contained phosphatidylcholine, cholesterol, and anionic phospholipids (phosphatidylserine, phosphatidic acid, or cardiolipin) that dramatically enhance the clearance rate of liposomes from the circulation. These anionic phospholipids are normally found exclusively in the interior of cells but become expressed when cells undergo apoptosis or programmed cell death, and thus, they are believed to be markers of cell senescence. Analysis of the proteins associated with these liposomes by SDS-polyacrylamide gel electrophoresis revealed that two of the major proteins associated with the liposome membranes are proteins with electrophoretic mobilities corresponding to M(r) of 66,000 and 50,000-55,000. The 66-kDa protein was identified to be serum albumin by immunoblot analysis. Using various biochemical and immunological methods, we have identified the 50-55-kDa protein as the murine equivalent of human beta 2-glycoprotein I. beta 2-glycoprotein I has a strong affinity for phosphatidylserine, phosphatidic acid, and cardiolipin inasmuch as the levels of beta 2-glycoprotein I associated with these anionic liposomes approach or even exceed those of serum albumin, which is present in serum at a concentration 200-fold greater than beta 2-glycoprotein I. Further, we demonstrate that the amount of beta 2-glycoprotein I associated with liposomes, as quantitated by an enzyme-linked immunosorbent assay, is correlated with their clearance rates; moreover, the circulation residency time of cardiolipin-containing liposomes is extended in mice pretreated with anti-beta 2-glycoprotein I antibodies. These findings strongly suggest that beta 2-glycoprotein I plays a primary role in mediating the clearance of liposomes and, by extension, senescent cells and foreign particles.

A two-step targeting approach was used to deliver doxorubicin-loaded liposomes to a murine tumour cell (P388 leukaemia) grown in culture and, more importantly, in vivo. Targeting was mediated through the use of an antibody specific for the Thy 1.2 antigen that is highly expressed on P388 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-Thy 1.2 antibody prior to administration of drug-loaded liposomes that have streptavidin covalently attached to their surface. Results from in vitro studies demonstrate that a 30-fold increase in cell-associated lipid and a 20-fold increase in cell-associated doxorubicin can be achieved over control liposomes using this two-step procedure. Flow-cytometry and fluorescent-microscopy data were used to confirm that P388 cells can be stably labeled with the biotinylated anti-Thy 1.2 antibody in vivo. Subsequently, liposome-targeting studies were initiated in vivo, where target cell binding was assessed following i.p. or i.v. injection of doxorubicin-loaded liposomes into animals bearing P388 tumours prelabeled with biotinylated antibody. A streptavidin-mediated 3.7-fold increase in cell-associated lipid and drug was achieved when the liposomes were given i.p. When doxorubicin-loaded streptavidin liposomes were injected i.v., P388 cells located in the peritoneal cavity were specifically labeled, although the efficiency of this targeting reaction was low. Less than a 2-fold increase in cell-associated lipid was achieved through the use of target-specific (streptavidin-coated) liposomes. These studies demonstrate that the presence of a well-labeled target cell population within the peritoneal cavity will not promote accumulation of an i.v. injected, targeted liposomal drug. Furthermore, the importance of separating target-cell-specific binding from non-specific uptake by tumour-associated macrophages is discussed.

Publisher Summary Biological membranes are fluid (liquid–crystalline) lipid bilayers, into which, proteins can insert or associate at the surface. The membrane research has focused primarily on the protein components, with the lipid portion viewed as a convenient barrier and environment for enzymes. However, biological membranes contain a wide diversity of lipids, far more than are needed to perform structural functions, and these lipids require elaborate metabolic pathways for their synthesis and transport. The main classes of lipids found in eukaryotic biological membranes include the glycerophospholipids, the sphingolipids, and cholesterol (Chol). Of the former group, phosphatidylcholine (PC) is the major lipid, but phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and cardiolipin (CL) are also major lipid species in biological membranes. A representative chemical structure of a common phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The sphingolipids include sphingomyelin (SPM), ceramide (CER), and the glycosphingolipids (GSLs). Most biological membranes are fluid at physiological temperatures—a requirement for proper function. The fluid-membrane phase usually refers to the liquid–crystalline bilayer phase, although membranes that contain large quantities of Chol can adopt a different fluid-phase known as liquid-ordered.

Prostate cancer is the most commonly diagnosed non-skin cancer in men and one of the leading causes of cancer-death. While frequently curable by surgical or radiation ablation, recurrent or metastatic disease is usually managed with androgen withdrawal therapies (medical castration), which block the growth promoting effects of androgens on the androgen receptor (AR). Unfortunately, the cancers eventually progress to the lethal castration-resistant state. Previously, we reported that knocking down the AR by RNA interference using small hairpin RNA (shRNA) inhibits this progression and, in about 50% of tumours, causes complete regression. In this study, we investigated the ability of small interfering RNA (siRNA) encapsulated in lipid nanoparticle (LNP) to silence AR in human prostate cancer cell lines and xenograft tumours following intravenous injections. In vitro screening studies with prostate cancer cell lines using a panel of cationic lipids demonstrated that siRNA formulated in LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to enter into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, we found that LNP AR-siRNA systems containing DLin-KC2-DMA could silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following intravenous injection. The 5′RACE experiments further confirmed that siRNA-induced specific cleavage of AR mRNA was the primary mechanism of action. In conclusion, these results indicate that in vivo delivery of siRNA to knockdown AR using our LNP formulations causes a decrease in serum PSA and provides proof in principle that this approach may offer a therapeutic strategy for treating advanced prostate cancer. (Funded by Canadian Institutes for Health Research and a PCF-STAR Project from Prostate Cancer Canada with the support of Safeway Canada) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1970. doi:1538-7445.AM2012-1970

Previous work [Chakrabarti et al. (1992) Biophys. J. 61, 228-234] has shown that basic amino acids and peptides, in which the C-terminal carboxyl groups have been modified to form amides or methyl esters, can be rapidly and efficiently accumulated into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients (delta pH, inside acidic). In this work, the ability of small (di and tri) peptides, composed exclusively of basic (lysine) and hydrophobic (tryptophan) amino acids, to accumulate into LUV systems in response to delta pH has been investigated. In the case of the dipeptides Trp-Lys-amide and Lys-Trp-amide, remarkable differences in the rate constants associated with net transport were observed. In EPC:cholesterol LUV systems exhibiting a delta pH of 3 units (pHi = 4.0; pHo = 7.0), for example, the rate constant for the uptake of Lys-Trp-amide is some 5 x 10(3) faster than for Trp-Lys-amide. Activation energies associated with the uptake also varied from 24 (Lys-Trp) to 29 kcal/mol (Trp-Lys). Related effects were observed for the tripeptides composed of one lysine and two tryptophan residues; however, the differences in rate constants were less sensitive to amino acid sequence. It is concluded that different charge distributions in short peptides of identical amino acid composition can strongly influence the ability of these groups to associate with and permeate across lipid bilayers. These observations may have relevance to the ability of basic peptides, such as signal sequences and peptide hormones, to translocate across biological membranes.

The Ca2+ translocating properties of the carboxylic ionophores A23187, ionomycin and lasolocid A (X537A) have been investigated by employing large unilamellar vesicles that exhibit a pH gradient (acidic interior). An analysis of Ca2+ uptake at equilibrium reveals that Ca2+ accumulation is an electroneutral process, whereby one Ca2+ ion is transported in for every two H+ ions transported out. A kinetic analysis shows that both A23187 and ionomycin transport Ca2+ in the form of a 1:1 cation:ionophore complex, whereas a 1:2 complex is observed for lasolocid A. The specificity of the ionophores for transporting Ca2+ is reflected by the influence of exterior Na+ ions that inhibit Ca2+ uptake for lasolocid A but do not influence ionomycin-mediated uptake.

Serum complement opsonizes particles such as bacteria for clearance by the reticuloendothelial system. Complement has been reported to interact with liposomes and therefore may mediate the reticuloendothelial system clearance of liposomes. This study has used a rat serum model to define some of the characteristics of liposomes which modulate their ability to activate complement. Using functional hemolytic assays and C3/C3b crossed immunoelectrophoresis, we have demonstrated that liposomes activated rat complement in a dose-dependent manner with higher concentrations of liposomes activating higher levels of complement. The detection of complement activation required the inclusion of phospholipids bearing a net charge. Complement activation occurred via the classical pathway; no alternative pathway activation was detected. The presence of cholesterol contributed to complement activation in a dose-dependent manner. Phospholipid fatty acyl chain length did not influence complement activation while the introduction of unsaturated acyl chains markedly decreased levels of complement activation. Liposome size also influenced complement activation with 400 nm unilamellar vesicles more effectively activating complement than 50 nm vesicles for equivalent amounts of exposed lipid. These studies demonstrate that the composition of the liposome greatly affects the in vitro activation of rat serum complement and suggest that the biological half-life of liposomes in the circulation of rats may be altered by changing the liposome composition to reduce complement activation.

Alterations in the lipid composition of biological membranes can have dramatic effects on their ability to interact with soluble proteins. In a series of studies employing large unilamellar vesicles (LUVs) produced by an extrusion technique, we have characterized the influence of membrane components on protein-membrane interactions. Much of our understanding of how and why proteins interact with seemingly inert membrane surfaces stems mainly from studies involving one or two protein component systems. These studies, however, do not accurately reflect the interactions that occur in the complex biological milieu (reviewed by Horbett and Brash, 1987). There have been very few studies reported on the interactions of proteins with liposomal systems incubated with whole blood. There are two main reasons for this. First, the large majority of studies on the association of plasma proteins with liposomes in vitro have been performed employing multilamellar systems. Due to the variable lamellarity of liposomes of different lipid compositions, quantification of the amount of various proteins associated per liposome has not been possible. Second, convenient techniques have not been available for the isolation of liposomes, particularly LUVs, from blood components. We have recently described a rapid method for the isolation of well-defined large unilamellar liposomes from the blood of liposome-treated mice (Chonn et al., 1991). With such a procedure now available, we have started to biochemically and immunologically characterize the amount and type of proteins associated with liposomes exposed to the complex biological milieu.

Previously we have demonstrated that the L1210 antitumor activity of liposomal doxorubicin increased significantly as the size of the liposomal carrier was reduced from 1.0 to 0.1 micron. It is demonstrated herein that empty and drug-loaded small (0.1-micron diameter) liposomes accumulate efficiently into the peritoneal cavity of normal and ascitic L1210 tumor-bearing animals following i.v. administration. In normal mice injected with 100 nm DSPC/chol liposomal doxorubicin (drug-to-lipid ratio of 0.2; wt/wt) approximately 2.8 micrograms drug could be recovered from the peritoneal cavity following peritoneal lavage at 24 h. Although this represents only 0.7% of the injected doxorubicin dose, this level of drug is 2 orders of magnitude greater than that achieved following administration of an equivalent dose of free drug (20 mg/kg). The drug levels achieved within the peritoneal cavity are dependent on the physical characteristics (size, drug-to-lipid ratio and lipid composition) of the liposomes employed. Optimal delivery is obtained employing 100 nm DSPC/chol liposomal doxorubicin, a vesicle system that is known to retain entrapped drug following i.v. administration and exhibits extended circulation lifetimes. Analysis of drug and liposome distribution within the peritoneal cavity of normal mice indicates that as much as 50% of the measured doxorubicin and liposomal lipid is cell-associated. Flow cytometric analysis of the peritoneal cells demonstrated that cell-associated doxorubicin resides almost exclusively within resident peritoneal macrophages. The increased delivery of doxorubicin to the peritoneal cavity of normal mice following i.v. administration of small (0.1-micron) liposomal doxorubicin is correlated with a pronounced (90%) and prolonged (14-day) suppression of resident peritoneal cells. Liposomal drug accumulation increased dramatically in animals with an established L1210 ascitic tumor. More than 5% of the injected dose was found in the peritoneal cavity of these animals 24 h after treatment with DSPC/chol liposomal doxorubicin as compared with a value of 0.03% of the injected dose achieved with free drug. It is proposed that accumulation of liposomes into the peritoneal cavity of normal and tumor-bearing mice may serve as a useful model for characterizing factors mediating the transfer of liposomes from the vascular compartment to extravascular sites.

The influence of lipid composition, internal pH and internal buffering capacity on the retention properties of vincristine loaded into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been assessed. It is shown that increasing the (saturated) acyl chain length of the phosphatidylcholine molecule, increasing the internal buffering capacity, and decreasing the internal pH all result in increased drug retention. Further, a study of the pH dependence on the rates of accumulation indicate that uptake proceeds via the neutral form of the vincristine molecule. This uptake is associated with an activation energy of 37 kcal/mol for DSPC/Chol LUVs. It is shown that the major improvement in drug retention in vitro is achieved by employing low initial internal pH values, where 90% retention is obtained over 24 h for an initial internal pH of 2. Improved retention in vivo was also observed where a drug-to-lipid ratio approx. 4-fold greater at 24 h was maintained.

Large unilamellar vesicles (LUVs) composed of 1-[2H31]palmitoyl-2-oleoyl phosphatidylcholine (POPC-d31), with diameters of approximately 117 +/- 31 and 180 +/- 44 nm, were prepared by extrusion through polycarbonate filters with pore sizes of 0.1 and 0.2 microns, respectively. The 2H nuclear magnetic resonance (NMR) spectra obtained at 21 degrees C contain two components: a broad component (approximately 17 kHz linewidth) corresponding to the methylene groups and a narrower component originating from the methyl groups. Spectra with increasing powder pattern characteristics were obtained by reducing the rate of phospholipid reorientations by addition of glycerol (to increase the solvent viscosity) and by lowering the temperature. Full powder spectra, characteristic of liquid-crystalline bilayers, were obtained for both LUV samples at 0 degrees C in the presence of 50 wt% glycerol. Individual quadrupolar splittings were not resolved in these spectra, due to broader linewidths in the LUVs, which have significantly shorter values for spin-spin relaxation time T2 measured from the decay of the quadrupolar echo (90 microseconds) than the multilmellar vesicles (MLVs; 540 microseconds). Smoothed order parameter profiles (OPPs) were obtained for these samples by integration of the dePaked spectra. The OPPs were very similar to the OPP of POPC-d31 MLVs in 50 wt% glycerol at the same temperature, indicating that orientational order in MLVs and LUVs with a diameter of > or = 100 nm is essentially the same. The presence of 80 wt% glycerol was found to have a disordering effect on the vesicles.

ABSTRAC T: Recent investigations have indicated that there exists a well-defined range of membrane hydrocarbon order compatible with good growth of the microorganism Acholeplasma laidlawii B [Monck, M., Bloom, M., Lafleur, M., Lewis, R. N. A. H., McElhaney, R. N., & Cullis, P. R. (1992) Biochemistry 31, 10037-10043]. Since cholesterol increases hydrocarbon order in membranes, it was of interest to examine the effect of cholesterol on the hydrocarbon order and growth characteristics o f A. laidlawii B. Cholesterol is normally absent fro m A. Zaidlawii membranes since it is neither biosynthesized nor required for the growth or survival of the microorganism .However, cholesterol will be incorporated into the membrane if exogenously supplied to the A. laidlawii culture. For membranes prepared from cells grown in the presence of cholesterol, chemical determinations indicated cholesterol represented as much as 40 mol % of the total membrane lipid .However, 2H NMR order parameter measurements and DSC studies of the same membrane preparation suggested that cholesterol was present at significantly lower levels (alOmol %) in the membrane lipid bilayer .Further incorporation of cholesterol into th e A. Zaidlawii lipid bilayer was found to occur with an increase in temperature or by lyophilization and rehydration at high temperatures, suggesting that sterol present in a separate pool in the membrane preparation could then gain access to the bilayer. 2H NMR spectra of A. laidlawii membrane preparations containing deuterium-labeled cholesterol indicate that the bulk of the cholesterol present in this separate pool is in a solid form.

The influence of vesicle lipid composition, size and drug-to-lipid ratio on the antitumour activity of liposomal vincristine was assessed in the murine L1210 ascitic leukemia model. A pH gradient-dependent entrapment procedure was used to encapsulate vincristine and allowed such vesicle properties to be independently varied. Free vincristine delivered i.v. at the maximum tolerated dose (2.0 mg/kg) resulted in a 27.8% increase in the life span (ILS) of mice inoculated i.p. with L1210 cells. Encapsulation of the drug in egg phosphatidylcholine/cholesterol vesicles did not significantly increase the antitumour efficacy of vincristine (ILS, 38.9%). In contrast, administration of vincristine entrapped in vesicles composed of distearoylphosphatidylcholine (DSPC)/cholesterol resulted in ILS values as high as 133%. This enhanced antitumour activity of the DSPC/cholesterol formulations was sensitive to the size of the liposomes; increasing the vesicle size from 100 nm to 1 micron decreased the ILS from 133.3% to 55.6% at a drug dose of 2.0 mg/kg. Decreasing the drug-to-lipid ratio from 0.1:1 to 0.05:1 (w/w) had negligible effects on the activity of liposomal vincristine; however, a further decrease in the drug-to-lipid ratio to 0.01:1 (w/w) decreased the antitumour potency at all drug doses studied. Pharmacology studies indicated that the antitumour activities of free and various liposomal forms of vincristine correlated well with the residence time of the drug in the circulation. These studies indicate that efforts to enhance the therapeutic activity of vincristine through liposome encapsulation must address not only the circulation lifetime of the vesicle systems but also the capacity of the liposomes to retain entrapped drug in vivo.

2H NMR techniques have recently been developed to determine the complete orientational order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid [Lafleur, M., Fine, B., Sternin, E., Cullis, P.R., & Bloom, M. (1989) Biophys. J. 56, 1037-1041]. In this work, these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A. laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuteriated fatty acid of varying length and unsaturation. By variation of the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic, or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter, mean value of S, varies over the range 0.140-0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2H NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.

Cholesterol 3-sulphate (CS) is a component of the intercellular lipid found in the uppermost layer of human epidermis (the 'stratum corneum') and is thought to play an important role in tissue cohesion. In this investigation we have compared the influence of cholesterol (CH) and CS on the gel-to-liquid crystalline phase behaviour, the polymorphic phase behaviour, and the hydrocarbon order profile in selected model membranes. It is shown that in sphingomyelin (SPM) systems, the presence of equimolar amounts of either CH or CS eliminates the gel-to-liquid crystalline transition as detected by calorimetry. Similarly, in 1-palmitoyl,2-oleoyl-phosphatidylethanolamine (POPE) dispersions containing a perdeuterated palmitoyl chain (POPE-d31), it is shown that both CH and CS exert an ordering effect as determined by 2H-NMR techniques, however, CS is less potent at temperatures both above and below that of the main transition for the native phospholipid. Alternatively, in mixed systems containing dioleoylphosphatidylethanolamine (DOPE) and SPM (DOPE/SPM, 6:1 mol/mol) CH promotes thermotropic L alpha-->HII phase transitions, whereas CS stabilizes the bilayer organization. These bilayer stabilization effects can be diminished by addition of Ca2+. These effects are consistent with a larger area per molecule of CS as compared to CH, presumably related to the presence of the negatively charged sulphate moiety of CS.

Previous work (Veiro and Cullis (1990) Biochim. Biophys. Acta 1025, 109-115) has shown that Ca2+ can be accumulated into large unilamellar vesicles (LUVs) in the presence of a transmembrane pH gradient (inside acidic) and the Ca(2+)-ionophore A23187. Here, the ability of A23187 to mediate the uptake of iron and barium into LUVs has been investigated. It is shown that under appropriate conditions of temperature and A23187 concentration, iron (in the form of Fe2+) can be accumulated into EPC and DSPC/cholesterol (55:45; mol/mol) LUVs with an acidic interior. This uptake is dependent on the internal buffer concentration, with maximum levels of uptake in the range of 300 nmol of cation per mumol lipid. The DSPC-cholesterol LUV systems exhibit superior retention properties compared to the EPC systems. It is demonstrated that Ba2+ can also be loaded by similar methods. It is also shown that the maximally loaded Fe(2+)- and Ba(2+)-containing LUVs exhibit increased densities. This is expressed by enhanced gravimetric properties, as an increased proportion of the loaded LUVs can be pelleted by low speed centrifugation, and by enhanced electron densities, in that the Ba(2+)-loaded systems can be directly visualized employing cryo-electron microscopy.

Preliminary studies have shown that asymmetric transbilayer distributions of phosphatidic acid (PA) can be induced by transmembrane pH gradients (delta pH) in large unilamellar vesicles [Hope et al. (1989) Biochemistry 28, 4181-4187]. Here the mechanism of PA transport is examined employing TNS as a fluorescent probe of lipid asymmetry. It is shown that the kinetics of PA transport are consistent with the transport of the uncharged (protonated) form. Transport of the neutral form can be rapid, exhibiting half-times for transbilayer transport of approximately 25 s at 45 degrees C. It is also shown that PA transport is associated with a large activation energy (28 kcal/mol) similar to that observed for phosphatidylglycerol. The maximum induced transbilayer asymmetry of PA corresponded to approximately 95% on the inner monolayer for vesicles containing 5 mol % PA.

Publisher Summary Membrane lipids satisfy demands related to membrane structure, fluidity, and permeability as well as protein association and function. This chapter describes the physical properties of lipid systems and indicates how they may relate to the functional capacities of biological membranes. The chapter also provides a basic overview of lipid diversity in membranes, a study of the methods employed to isolate individual components, and a discussion of the physical properties of lipids. Although, the physical properties of membrane lipids are well understood, the relation between these physical properties and the functional roles of lipids remain relatively obscure. The ability of certain classes of lipids to adopt nonbilayer structures are probably of utility in inter-membrane interactions, such as fusion, which require local departures from the bilayer organization. These abilities can be satisfied by a relatively limited subset of the lipids actually present in membranes. Important additional functions, which require clarification, include the detailed roles of lipids in establishing and modulating membrane permeability and membrane protein function as well as their roles in transbilayer signaling phenomena.

The orientational order profile has been determined by using deuterium nuclear magnetic resonance (2H NMR) for POPE in the lamellar liquid-crystalline (La) and the hexagonal (HII) phases and is shown to be sensitive to the symmetry of the lipid phase. In the HII phase, as compared to the La phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. *H NMR studies of POPE dispersions containing tet- radecanol or decane, both of which can induce HII phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the La. to HII phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The La to HII phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. This is consistent with a preferential partition of the decane molecules into a disordered environment such as the intercylinder spaces in the HI1 phase. The presence of decane in the HII phase structure does not modify the order of the lipid chains. However, the La phase of POPE is slightly disordered by the addition of 9 mol % decane whereas it can accommodate as much as 20 mol 76 tetradecanol without a significant change of order. Finally, the concept of a stretching vector associated with the lipid acyl chain has been introduced to analyze the orientational order profile obtained in the HII phase. With this model, the average order parameter of the HI1 phase has been calculated and found to be in good agreement with experiment.

Tumor drug uptake and antitumor efficacy of free and liposomal doxorubicin (DOX) were determined in the SC115 Shionogi mouse mammary tumor. Liposomal DOX systems were prepared by pH gradient-driven drug encapsulation in 170 nm egg phosphatidylcholine/cholesterol (55:45, mol ratio) vesicles. Intravenous injection of free DOX at 6.5 mg/kg, the maximum tolerated dose for free drug in the multiple dose therapy regimen, resulted in tumor-associated drug levels of 2.0 micrograms/g tissue at 1 h which remained constant over 24 h. Liposomal DOX injected at 6.5 mg/kg led to an accumulation of drug in the tumor from 2.6 micrograms/g tissue to 5.5 micrograms/g tissue between 1 h and 24 h, respectively. Increasing the dose of liposomal DOX to 13.0 mg/kg increased tumor drug uptake levels to 5.7 micrograms/g and 10.2 micrograms/g tissue at 1 h and 24 h, respectively. Administration of free or liposome encapsulated DOX every 7 days for 3 weeks resulted in a dose-dependent decrease in tumor growth rate. However, liposomal DOX injected at 6.5 mg/kg exhibited enhanced tumor growth inhibition compared to an equivalent dose of free drug. Further, the ability to administer increased doses of the less toxic liposomal DOX not only resulted in a greater inhibition of tumor growth but also significantly reduced tumor weight. Tumors weighing as much as 5 g were diminished to less than 0.5 g upon treatment with liposomal DOX at a dose of 13 mg/kg. In addition, groups receiving the highest liposomal DOX dose exhibited 25% complete tumor regression which persisted over the 50-day study period. These results demonstrate the ability of appropriately designed liposomal DOX systems to significantly enhance the delivery and retention of drug at solid tumor sites, resulting in increased therapeutic activity.

A technique for the efficient entrapment of high concentrations of Ca2+ in large unilamellar phospholipid vesicles (LUVs), using the carboxylic acid antibiotic ionophore A23187 (calcimycin) is demonstrated. It is shown that rapid A23187-mediated entrapment of Ca2+, corresponding to essentially 100% sequestration of the extravesicular cation may be achieved for egg yolk phosphatidylcholine LUVs (100 nm) in the presence of a transmembrane proton gradient (acidic interior). Interior-exterior concentration cation gradients of over 400-fold may be readily achieved, with interior Ca2+ concentrations in excess of 250 mM. It is shown that the extent and efficiency of the A23187-mediated uptake process is affected by the intravesicular buffering capacity and the extravesicular Ca2+ concentration in a manner that is consistent with a Ca2(+)-H+ exchange process. In the absence of a pH gradient, or the presence of a reversed gradient (basic interior), only background levels of cation uptake are detected. The driving force for A23187-mediated uptake of Ca2+ is shown to depend on the intravesicular proton pool rather than on a chelation process. This protocol provides a novel method for the efficient entrapment of high concentrations of Ca2+ and other cations in phospholipid vesicles.

Previous work [Hope et al. (1989) Biochemistry 28, 4181-4187] has shown that asymmetric transmembrane distributions of phosphatidylglycerol (PG) in PG-phosphatidylcholine (PC) large unilamellar vesicles can be induced in response to transbilayer pH gradients (delta pH). Here the mechanism of PG transport has been investigated. It is shown that PG movement in response to delta pH is consistent with permeation of the uncharged (protonated) form and that the half-time for transbilayer movement of the uncharged form can be on the order of seconds at 45 degrees C. This can result in rapid pH-dependent transmembrane redistributions of PG. The rate constant for transbilayer movement exhibits a large activation energy (31 kcal/mol) consistent with transport of neutral dehydrated PG where dehydration of the (protonated) phosphate presents the largest barrier to transmembrane diffusion. It is shown that acyl chain saturation, chain length, and the presence of cholesterol modulate the rate constants for PG transport in a manner similar to that observed for small nonelectrolytes.

We have shown previously that transmembrane proton gradients can be used to efficiently accumulate biogenic amines [M.B. Bally et al. (1988) Chem. Phys. Lipids 47, 97-107] and doxorubicin [L.D. Mayer, M.B. Bally and P.R. Cullis (1986) Biochim. Biophys. Acta 857, 123-126] to high concentrations within liposomes. To determine the generality of this loading procedure, representative drugs from a variety of different classes (antineoplastics, local anaesthetics, antihistamines, etc.) were examined as to their ability to redistribute in response to a proton gradient. While the majority of drugs examined, all of which are weak bases, were accumulated by large unilamellar vesicles exhibiting a pH gradient (interior acid) the extent of uptake varied considerably between different pharmaceuticals. These differences are discussed in the context of various factors which will likely influence drug accumulation including its membrane/water partition coefficient and its solubility in the intravesicular medium.

The use of 2H nuclear magnetic resonance for the characterization of the polymorphic behavior of lipids is illustrated. Different lipid phase preferences may be expected to influence the orientational order and its variation along the acyl chains. Several results are presented to support that view. An increase of motional freedom and a redistribution of the order along the acyl chains are observed during the lamellar-to-hexagonal phase transition, showing that the order profile is sensitive to the lipid phase symmetry. In addition, if the preferences for nonlamellar phases are not expressed explicitly, the presence of "nonbilayer" lipids constrained in bilayer environment induces increased hydrocarbon order. This suggests that order parameters of the acyl chains and lipid polymorphic tendencies are intimately related.Key words: lipid, polymorphism, 2H nuclear magnetic resonance, hydrocarbon order.

The orientational order profile along the lipid acyl chain has been characterized under several different conditions of polar headgroup composition, temperature, and cholesterol content. Despite the different nature of these factors, the variation of the order is governed by two common trends. First, the relative change of order induced by the variation of these factors is always more pronounced towards the end of the chain than for the methylene groups near the interface. Second, there is, to a first approximation, a distinct correlation between the magnitude of the order parameters and the shape of the order profile. For example when the chain is highly ordered, the relative width of the order distribution is narrow indicating that the plateau region is longer. These conclusions suggest that the orientational order profile depends on only a small number of parameters and demonstrate clearly that the correlation length for changes in orientational order is much greater than one C-C bond length. Our results also show that the reduced temperature is not related in simple terms to orientational order and probably has little theoretical significance. The orientational order profiles of POPC and POPE bilayers are significantly different even when expressed in terms of reduced temperature. The behavior of POPC/cholesterol systems also indicates that the orientational order of the lipid chain and the gel-to-liquid crystalline phase transition temperature are not related in a straightforward manner.

When liposomes are subjected to dehydration or freeze-thawing, vesicle fusion and/or leakage of vesicle contents can occur. The disaccharide, trehalose and the cryoprotectant, glycerol, are known to protect vesicle integrity during dehydration and freezing respectively. Here we examine their protective abilities as a function of vesicle size and lipid composition. It is shown that fatty acyl composition, cholesterol content and, with the exception of phosphatidylglycerol, acidic lipid content do not significantly alter the retention of aqueous contents by vesicles dehydrated and rehydrated in the presence of trehalose. The susceptibility to leakage induced by both dehydration and freezing is, however, critically dependent upon vesicle size with the smallest systems (70-100 nm diameter) being most stable. The mechanism whereby trehalose protects against vesicle fusion and leakage is also discussed.

The myelosuppressive activity of doxorubicin encapsulated in liposomes of differing lipid composition and size was quantified in mice by measurement of changes in spleen weight, peripheral white blood cells (WBC), and bone marrow nucleated cells. Following i.v. administration of free doxorubicin at a dose of 20 mg/kg, a 90% reduction in marrow cellularity was observed on day 3. The marrow nucleated cell count was similar to control values by day 7. Administration of an equivalent dose of doxorubicin that was encapsulated in large (diameter, approximately 1.0 microns) egg phosphatidylcholine/cholesterol (EPC/Chol)(molar ratio, 55:45) liposomes induced an 80% reduction in bone marrow cellularity that lasted for periods of greater than 7 days. Similar results were obtained following administration of large (1.0 microns) liposomal doxorubicin systems formulated with distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (molar ratio 55:45). In contrast, liposomal doxorubicin prepared using small (diameter, approximately 0.1 micron) DSPC/Chol liposomes induced only a 40% reduction (day 3) in bone marrow cellularity, which returned to control values by day 7. Other indicators of doxorubicin-mediated myelosuppressive activity (spleen weight loss and peripheral leukopenia) correlated well with changes observed in marrow cellularity. An exception to this, however, was observed in animals treated with small (0.1 -micron) DSPC/Chol Liposomal doxorubicin, which displayed peripheral leukopenia for periods of greater than 14 days. This extended leukopenia was not observed following administration of small (0.1 -micron) EPC/Chol liposomal doxorubicin. Marrow-associated liposomal lipid and doxorubicin were quantified to determine if the extent of doxorubicin-mediated myeloid toxicity could be correlated to changes in biodistribution of the entrapped drug. It was demonstrated that 10-20 times more doxorubicin is delivered to the bone marrow when the drug is given encapsulated in large liposomes than when it is associated with small liposomes. These data are useful in defining characteristics of liposomal preparations that modulate the myelosuppressive behaviour of entrapped antineoplastic agents.

Background: Vincristine sulfate (VCR) is a cell-cycle specific, lipophilic, anti-cancer drug that inhibits cell division by binding to tubulin in mitotic spindles. Marqibo is a proprietary sphingomyelin/cholesterol liposome (OPTISOME) encapsulated formulation of VCR with an extended circulating half-life and the potential for preferential malignancy targeting, exposure, and anti-cancer activity. This report evaluates the concentration and accumulation of VCR in solid tumor tissue as well as tissues frequently involved with lymphoid malignancies in tumor-bearing mice following equivalent doses of Marqibo or conventional VCR. Methods: Mice were implanted subcutaneously with MX-1 human breast tumor tissue. When the tumor volume reached 162–485 mm3, mice received a single intravenous dose of Marqibo, containing 1.5 mg/kg of [14C]-VCR, or 1.5 mg/kg of non-liposomal [14C]-VCR. The total radioactivity from parent compound and metabolites in tissue, VCReq, was analyzed by Quantitative Whole Body Autoradioluminography (QWBA) and tissue digestion (TD). QWBA analysis of bone marrow (BM), tumor tissue, lymph nodes, liver, and spleen required 1 flash-frozen mouse per group at various time intervals. Sagittal sections were examined histologically for radioactivity. For TD, all tissues except bone marrow were obtained from 3 mice per group prior to infusion and at selected time points. The samples were chemically digested and analyzed for radioactivity by liquid scintillation counting. Results: QWBA analysis revealed at least two-fold higher VCReq tissue concentrations in the Marqibo-injected mice compared to the conventional VCR-injected mice. | | VCReq Tissue Concentration (mcg/g) at 48 hours Post-Injection | |:-----------------:| ------------------------------------------------------------- | ----- | | | BM | Tumor | Lymph | Liver | Spleen | | Marqibo | 0.99 | 1.35 | 1.98 | 0.47 | 6.08 | | VCR | 0.36 | 0.31 | -- | 0.13 | 0.76 | | Marqibo/VCR ratio | 2.75 | 4.35 | -- | 3.61 | 8 | The TD results are consistent with the QWBA results shown in the table. Marqibo-injected mice demonstrated a minimum 5-fold increase in VCR exposure over a 96 hour period (VCReq AUC analysis) compared to VCR-injected mice over a 48 hour period, as defined in the parameters of the study, in the implanted tumor (123 vs. 21 hr*μg/g), lymph nodes (121 vs. 16.5 hr*μg/g), liver (68.8 vs. 12 hr*μg/g), and spleen (512 vs. 68.9 hr*μg/g). Though the parameters for the AUC values may favor Marqibo, the Tmax of all specified tissues are at least 16 times greater in Marqibo-injected mice than in VCR-injected mice (16 hrs vs. 1hr in tumor, spleen, and lymph; 4 hrs vs. 15 min. in liver; 16 hrs. vs. 15 min. in the kidney). The greatest increase in VCR exposure produced by Marqibo was observed in the spleen and lymph nodes (7.4- and 7.3-fold). Conclusions: Marqibo resulted in targeted delivery of VCR; concentration of VCR in tumor tissue, BM, lymph nodes, liver, and spleen; and maintenance of significant tissue drug concentrations for an extended period of time compared to conventional VCR. The ability of Marqibo to target drug to these tissues and organs makes it particularly attractive as a treatment for hematologic malignancies like myeloma, lymphoma, and acute lymphoblastic leukemia (ALL). Late-stage clinical development of Marqibo in adult ALL is underway.

Every organelle in a eukaryotic cell contains enzymes with specialized functions, unique to that organelle. But the endosome does things differently — it is regulated, in part, by a unique lipid called lysobisphosphatidic acid (LBPA). This lipid seems to be crucial, because anti-LBPA antibodies affect both the structure and function of the late endosome. Moreover, LBPA is recognized by autoimmune sera from patients with antiphospholipid syndrome, indicating that it may be involved in pathogenesis of this disease.

Background Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line) and COS-7 (a low expressing cell line). Results The main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence) are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes. Conclusion After exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.

The polymorphic phase behavior of mixtures of synthetic dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) and the influence of cholesterol on these phase preferences have been investigated by employing nuclear magnetic resonance (NMR) techniques. In particular, 31P NMR procedures are utilized to study the overall phase preferences of these mixed systems, whereas 2H NMR is employed to monitor the structural preferences of individual components of these systems by using versions of DOPE and DOPC which are deuterium (2H) labeled at the C11 position of the acyl chains. The results obtained show that DOPE-DOPC systems containing as little as 20 mol % DOPC initially assume lamellar structure at 40 degrees C, even though DOPE in isolation prefers the hexagonal (HII) organization at this temperature. However, this lamellar organization appears to represent a metastable state, as incubation for extended periods at 40 degrees C results in formation of a structure, possibly the cubic phase, in which the phospholipids experience isotropic motional averaging. The addition of cholesterol induces hexagonal (HII) phase organization. 2H NMR studies of appropriately labeled versions of these systems indicate that cholesterol does not produce such effects by associating preferentially with either DOPE or DOPC. Further, in situations where bilayer, hexagonal, or "isotropic" phases coexist in the same sample, the phospholipids exhibit apparently ideal mixing behavior.

Local amine anesthetics appear to exert their effects in the charged (protonated) form on the cytoplasmic side of excitable membranes. Two features of interest are the mechanism whereby these drugs move across the membrane to the inner monolayer and the actual membrane concentrations achieved. In this work, we have investigated the influence of a K+ diffusion potential, delta psi, on the transmembrane distribution and concentration of the local anesthetic dibucaine employing large unilamellar vesicle systems. It is demonstrated that egg phosphatidylcholine large unilamellar vesicles exhibiting a delta psi (interior negative) actively accumulate dibucaine to achieve high interior concentrations. 31P and 13C nuclear magnetic resonance studies show that the internalized drug is localized to the vesicle inner monolayer, and suggest that the protonated form of the anesthetic is the species that is actively transported. The inner monolayer anesthetic concentrations thus achieved can be an order of magnitude or more larger than predicted on the basis of anesthetic lipid-water partition coefficients. It is suggested that these effects may be related to the mechanisms whereby local anesthetics are localized and concentrated at their sites of action in nerve membranes.

SummaryThis investigation determined the organ distribution of liposomes containing egg phosphatidylcholine and cholesterol with egg phosphatidylglycerol (PG liposomes) or without (PC liposomes) and the effect of each liposome on platelet distribution in rabbits. Eight minutes after51 chromium-labelled platelets were given intravenously, either saline (n = 7), iodinated PG liposomes (n = 5) or iodinated PC liposomes (n = 5) were infused. T\ro minutes later the organ distribution of 51Cr-platelets and 125I-liposomes were compared. The PG liposomes produced a 41λ5% reduction in circulating platelet counts while PC liposomes did not. The PG liposomes decreased circulating 51Cr-platelets by a factor of 2 and increased platelet recoveries in the liver and lungs. The increased platelet recovery in the liver was associated with a greater PG liposome recovery. When animals receiving PG liposomes were studied over 60 minutes, both the labelled and unlabelled platelet counts returned to control values by 30 minutes and the 51Cr-platelet distribution between organs was similar to control values. These data indicate that platelets and PG liposomes initially sequester together and that this plateletliposome interaction is specific for PG liposomes. However, the platelet sequestration is transient and by 60 minutes the platelets were released and circulating.

Summary The structural preferences of saturated and unsaturated species of phosphatidylglycerol in isolation and in mixed systems with unsaturated (H II phase) phosphatidylethanolamine have been investigated employing 31 P NMR techniques. It is shown that equimolar (with respect to change) Ca 2+ induces a “rigid lattice” (no motion) situation in the phosphate region of the polar headgroup for a saturated species, but not for unsaturated species. Further, phosphatidylglycerol can stabilize a bilayer organization in the presence of phosphatidylethanolamine which would otherwise assume the hexagonal (H II ) phase, and structural bilayer-H II transitions can then be triggered in these systems by the addition of Ca 2+ .

1. 1. 31P NMR is a useful analytical technique for the study of the polymorphic phase behaviour of hydrated phospholipids in excess water. Such possibilities arise due to lateral diffusion of phospholipids which, in non-bilayer phases, produce additional motional averaging mechanisms. This results in distinctive 31P NMR spectra for lipids in the bilayer phase, hexagonal (H11) phase, or phases such as the inverted micellar, cubic or rhombic. 2. 2. The polymorphic phase behaviour of soya phosphatidylethanolamine and mixtures of soya phosphatidylethanolamine with saturated and unsaturated phosphatidylcholines has been investigated. As the temperature is decreased below −10°C soya phosphatidylethanolamine tends to enter the bilayer phase. At higher temperatures (0–50°C) the hexagonal (H11) phase is observed. The addition of 50 mol% egg yolk or (liquid-crystalline) 16 : 016 : 0 phosphatidylcholine is sufficient to stabilize the bilayer phase. 3. 3. The addition of equimolar cholesterol to 18 : 1c18 : 1c phosphatidylethanolamine results in the bilayer-hexagonal (H11) transition moving to lower temperatures. Similarly, the addition of equimolar cholesterol to soya phosphatidylethanolamine/35 mol% egg yolk phosphatidylcholine mixtures destabilizes the component in the bilayer phase. Alternatively, the addition of cholesterol to mixtures containing 30 mol% 16 : 016 : 0 phosphatidylcholine stabilizes the bilayer configuration. These results are discussed with regard to the observation that biological membranes containing high concentrations of phosphatidylethanolamine contain little or no cholesterol. Itis suggested that cholesterol may be excluded because its presence would disrupt bilayer structure. 4. 4. It is proposed that the bilayer structure of lipids in biomembranes may be in dynamic equilibrium with other available phases. In the light of such possibilities a mechanism of “flip-flop” phenomena in biological membranes is suggested.

31P-NMR shows that poly( l -lysine) binding to cardiolipin, phosphatidylserine or phosphatidylglycerol does not affect the macroscopic structure or local order (in the phosphate region) of the phospholipids. In the case of cardiolipin poly( l -lysine) inhibits the ability of Ca2+ to induce the hexagonal HII phase. Alternatively, poly( l -lysine) induces the hexagonal HII phase for a fraction of the phospholipids in phosphatidylethanolamine-cardiolipin (2:1) dispersions.

The motional properties of various well-defined species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, as well as phospholipids from natural sources, were investigated in oriented multibilayer systems employing 31P NMR. Particular hydrocarbon phase transition transition temperature Tc. The effective chemical shift anisotropy observed for hydrated phospholipids is relatively insensitive to the occurrence of gel-liquid-crystalline phase transitions, whereas the dipolar broadening experienced by the phosphate phosphorus in the nonproton decoupled spectra increases dramatically below Tc. It is shown that the basic features of the results obtained may be explained by a simple model where rapid axial rotation (τr ⪡ 10−5 sec) of the entire phospholipid molecule and rapid lateral diffusion occur above Tc, whereas below Tc the axial rotation remains, but the lateral diffusion is restricted. A theory is presented that accounts for the angular dependence of the linewidths of the 31P NMR spectra of the oriented lipid systems in the gel and liquid-crystalline phase, using a second-moment formalism.

We studied the ability of lipid-transfer factors in plasma to promote transfer, to endogenous lipoproteins, of [3H]cholesteryl ester from high-density lipoprotein (HDL) covalently bound to Sepharose 4B beads. After incubation for 2 h at 37 degrees C, 12 to 14% of the [3H]cholesteryl ester had been transferred to the lipoproteins of the plasma, in the proportions 57% to HDL and 43% to low- and very-low-density lipoproteins. This process was a function of the amount of plasma present and was stimulated by addition of partly purified lipid-transfer protein. Transfer also depended on the concentration of donor HDL but was independent of the amount of acceptor lipoprotein. This simple evaluation of cholesteryl ester transfer does not require removal of lipoproteins from the plasma before incubation.

The influence of temperature and ionic strength on the vesiculation properties of large multilamellar vesicles containing various proportions of dimyristoylphosphatidylglycerol has been investigated. It is shown that at low ionic strengths preformed large multilamellar vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3) on incubation at the gel to liquid-crystalline transition temperature (Tc approximately 23 degrees C) spontaneously vesiculate to form predominantly unilamellar systems with a mean diameter of 120 nm. Such vesiculation is not observed for incubations at temperatures appreciably above or below Tc, and is also inhibited by higher ionic strengths. Stable large multilamellar vesicles are formed, however, in systems containing the dioleoyl species of phosphatidylcholine or phosphatidylglycerol and also for dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine mixtures. The vesiculation properties of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol mixtures, therefore, appear to reflect an instability in the region of the Tc driven by surface potential effects which are specific for the glycerol headgroup.

The structural preferences of mixed lipid systems containing egg yolk or 18:1 c 18:1 c phosphatidylethanolamine and representative detergents (Triton X-100, deoxycholate, octylglucoside and lyso-phosphatidylcholine) have been examined. It is shown that all these detergents exhibit an ability to stabilize a bilayer organization for the phosphatidylethanolamine at detergent to phosphatidylethanolamine molar ratios of 0.05 to 0.5, depending on the detergent and/or phosphatidylethanolamine species. These results are interpreted in terms of molecular shape, where the ‘inverted cone’ shape detergents combine in a complementary fashion with ‘cone shaped’ phosphatidylethanolamine to result in net bilayer structure.