Biomedical subjects

P R Cullis

Publications and source records attributed to P R Cullis.

At least 55 records · Page 3Linked to original sources

PubMed1996

Correlation between lipid plane curvature and lipid chain order.

The 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE:POPC) system has been investigated by measuring, in the inverted hexagonal (HII) phase, the intercylinder spacings (using x-ray diffraction) and orientational order of the acyl chains (using 2H nuclear magnetic resonance). The presence of 20 wt% dodecane leads to the formation of a HII phase for the composition range from 0 to 39 mol% of POPC in POPE, as ascertained by x-ray diffraction and 2H nuclear magnetic resonance. The addition of the alkane induces a small decrease in chain order, consistent with less stretched chains. An increase in temperature or in POPE proportion leads to a reduction in the intercylinder spacing, primarily due to a decrease in the water core radius. A temperature increase also leads to a reduction in the orientational order of the lipid acyl chains, whereas the POPE proportion has little effect on chain order. A correlation is proposed to relate the radius of curvature of the cylinders in the inverted hexagonal phase to the chain order of the lipids adopting the HII phase. A simple geometrical model is proposed, taking into account the area occupied by the polar headgroup at the interface and the orientational order of the acyl chains reflecting the contribution of the apolar core. From these parameters, intercylinder spacings are calculated that agree well with the values determined experimentally by x-ray diffraction, for the variations of both temperature and POPE:POPC proportion. This model suggests that temperature increases the curvature of lipid layers, mainly by increasing the area subtended by the hydrophobic core through chain conformation disorder, whereas POPC content affects primarily the headgroup interface contribution. The frustration of lipid layer curvature is also shown to be reflected in the acyl chain order measured in the L alpha phase, in the absence of dodecane; for a given temperature, increased order is observed when the curling tendencies of the lipid plane are more pronounced.

Biophysical Phenomena↗

PubMed1996

Liposomal doxorubicin.

Doxorubicin is a potent antineoplastic agent with activity against numerous human cancers. Encapsulation of doxorubicin inside a liposome alters bioavailability, biodistribution and thus its biological activity significantly. The physical properties of the liposome (size, lipid components and lipid dose) play a major role in determining drug retention and pharmacokinetics. The therapeutic benefits of liposomal doxorubicin will therefore depend on these physical characteristics. Here we review the toxicity and efficacy of liposomal doxorubicin determined for various liposome compositions (size, lipid composition and drug-to-lipid ratio). These physical properties can be independently varied using the transmembrane pH gradient-dependent drug encapsulation procedure. The results show that the toxicity of the formulation is related to drug retention in the circulation. The antitumor activity is more sensitive to the size of the liposomes. By optimizing these parameters, liposomal doxorubicin formulations can be optimized for improved therapeutic activity.

Antibiotics, Antineoplastic↗

PubMed1995

Beta 2 glycoprotein I is a major protein associated with very rapidly cleared liposomes in vivo, suggesting a significant role in the immune clearance of "non-self" particles.

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.

Amino Acid Sequence↗

PubMed1995

A two-step targeting approach for delivery of doxorubicin-loaded liposomes to tumour cells in vivo.

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.

Animals↗

PubMed1995

Recent advances in liposomal drug-delivery systems.

Liposomal drug-delivery systems have come of age in recent years, with several liposomal drugs currently in advanced clinical trials or already on the market. It is clear from numerous pre-clinical and clinical studies that drugs, such as antitumor drugs, packaged in liposomes exhibit reduced toxicities, while retaining, or gaining enhanced, efficacy. This results, in part, from altered pharmacokinetics, which lead to drug accumulation at disease sites, such as tumors, and reduced distribution to sensitive tissues. Fusogenic liposomal systems that are under development have the potential to deliver drugs intracellularly, and this is expected to markedly enhance therapeutic activity. Advances in liposome design are leading to new applications for the delivery of new biotechnology products, such as recombinant proteins, antisense oligonucleotides and cloned genes.

Biotechnology↗

PubMed1995

Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles.

The morphological consequences of differences in the monolayer surface areas of large unilamellar vesicles (LUVs) have been examined employing cryoelectron microscopy techniques. Surface area was varied by inducing net transbilayer transport of dioleoylphosphatidylglycerol (DOPG) in dioleoylphosphatidylcholine (DOPC):DOPG (9:1, mol:mol) LUVs in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner to the outer monolayer, initially invaginated LUVs are transformed to long narrow tubular structures, or spherical structures with one or more protrusions. Tubular structures are also seen in response to outward DOPG transport in DOPC:DOPG:Chol (6:1:3, mol:mol:mol) LUV systems, and when lyso-PC is allowed to partition into the exterior monolayer of DOPC:DOPG (9:1, mol:mol) LUVs in the absence of DOPG transport. Conversely, when the inner monolayer area is expanded by the transport of DOPG from the outer monolayer to the inner monolayer of non-invaginated LUVs, a reversion to invaginated structures is observed. The morphological changes are well described by an elastic bending theory of the bilayer. Identification of the difference in relaxed monolayer areas and of the volume-to-area ratio of the LUVs as the shape-determining factors allows a quantitative classification of the observed morphologies. The morphology seen in LUVs supports the possibility that factors leading to differences in monolayer surface areas could play important roles in intracellular membrane transport processes.

Cholesterol↗

PubMed1995

Accumulation of protein-coated liposomes in an extravascular site: influence of increasing carrier circulation lifetimes.

The primary objective of this work was to test whether increased blood levels and circulation lifetimes result in increased passive targeting of protein-coated liposomal drug carriers. The system used to evaluate this was based on i.v. injection of 100 nm of distearoyl phosphatidylcholine/cholesterol liposomes with covalently bound streptavidin. The circulation lifetime of these liposomes was increased by procedures that involved blockade of liposome uptake by phagocytic cells in the liver and/or the incorporation of a poly(ethylene glycol)-modified phospholipid [poly(ethylene glycol)2000-modified distearoyl phosphatidylethanolamine]. Blockade of liver phagocytic cells with a low predose (2 mg/kg of drug) of liposomal doxorubicin increased the circulation half-life of the streptavidin liposomes from less than 1 hr to greater than 3 hr. A further 2-fold increase in circulating half-life (to approximately 7.5 hr) was achieved by using liposomes with 2 mole % of poly(ethylene glycol)2000-modified phosphatidylethanolamine. In combination with RES blockade, the circulation lifetimes of poly(ethylene glycol)phosphatidylethanolamine containing streptavidin liposomes could be increased to greater than 12 hr. The ability of these liposomes to move from the plasma compartment to an extravascular compartment was measured by using the peritoneal cavity as a convenient, accessible, extravascular site. The tendency for liposomes to accumulate in this site was not, however, clearly dependent on circulating blood levels. Comparable levels of liposomes in the peritoneal cavity were achieved when using systems that exhibited significantly different circulation lifetimes.

Animals↗

PubMed1994

Modulation of membrane fusion by asymmetric transbilayer distributions of amino lipids.

The fusion of model lipid bilayers containing synthetic amino lipids and the regulation of this fusion by inducing transbilayer asymmetry of these amino lipids via imposed pH gradients are demonstrated. Liposomes of 100 nm diameter consisting of 5 mol% 1,2-dioleoyl-3-(N,N-dimethylamino)propane (AL1) in a mixture of egg phosphatidylcholine (EPC), dioleoylphosphatidylethanolamine (DOPE), and cholesterol in a ratio of 35:20:45 do not fuse at pH 4.0. Fusion also is not observed upon increasing the external pH of these vesicles to 7.5, which results in the rapid transport of AL1 to the inner monolayer, as measured by a fluorescent probe sensitive to surface charge. However, dissipation of the imposed pH gradient leads to redistribution of AL1 to the outer monolayer at pH 7.5 and causes liposomal fusion, as detected by fluorescent lipid-mixing assay and freeze-fracture electron microscopy. The effect of varying the hydrocarbon structure of AL1 on the rate of fusion is demonstrated with five synthetic analogues, AL2-AL6. Higher rates of fusion occur with lipids containing longer unsaturated acyl chains and with lower values of pKa for the membrane-bound amino lipids. Fusion is also associated with destabilization of the bilayer at pH 7.5, as indicated by the formation of the hexagonal HII phase.

Fluorescent Dyes↗

PubMed1994

Factors influencing the retention and chemical stability of poly(ethylene glycol)-lipid conjugates incorporated into large unilamellar vesicles.

Poly(ethylene glycol)(PEG)-lipid anchor conjugates can prolong the circulation lifetimes of liposomes following intravenous injection. In this work we investigate the influence of the lipid anchor and the nature of the chemical link between the PEG and lipid moieties on circulation lifetime. It is shown that incorporation of N-(monomethoxypoly(ethylene glycol)2000-succinyl)-1-palmitoyl-2-oleoylphosphatidylethanolamide (MePEG2000-S-POPE) into large unilamellar vesicles (LUVs) composed of distearoylphosphatidylcholine (DSPC) and cholesterol (DSPC/cholesterol/MePEG2000-S-POPE, 50:45:5, mol/mol) results in only small increases in the circulation lifetimes as observed in mice. This is shown to be due to rapid removal of the hydrophilic coating in vivo, which likely arises from exchange of the entire PEG-lipid conjugate from the liposomal membrane, although chemical breakdown of the PEG-lipid conjugate is also possible. The chemical stability of four different linkages was tested, including succinate, carbamate and amide linkages between MePEG derivatives and the amino head group of PE, as well as a direct link to the phosphate head group of phosphatidic acid (PA). The succinate linkage was found to be the most labile. The anchoring capability of DSPE as compared to POPE in PEG-PE conjugates was also examined. It is shown that incorporation of MePEG2000-S-DSPE conjugates into DSPC/cholesterol LUVs results in little loss of the PEG coating in vivo, long circulation lifetimes and reduced chemical breakdown of the PEG-lipid conjugate. This work establishes that DSPE is a considerably more effective anchor for PEG2000 than POPE and that the chemical stability of PEG-PE conjugates is sensitive to the nature of the linkage and exchangeability of the PEG-PE complex. We suggest that retention of the PEG coating is of paramount importance for prolonged circulation lifetimes.

Animals↗

PubMed1994

Influence of charge, charge distribution, and hydrophobicity on the transport of short model peptides into liposomes in response to transmembrane pH gradients.

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.

Amino Acid Sequence↗

PubMed1994

Liposomal vincristine which exhibits increased drug retention and increased circulation longevity cures mice bearing P388 tumors.

Prolonged exposure to vincristine correlates with improved therapeutic activity. In this work, two methods are used to increase the circulation longevity of liposomal formulations of vincristine. The first involves incorporation of the ganglioside GM1, which acts to increase the circulation longevity of liposomal carriers, while the second approach relies on a modification of the vincristine encapsulation procedure which enhances drug retention. It is shown that these approaches are synergistic and increase the circulation half-life of vincristine from approximately 1 h to greater than 12 h. This results in a dramatic improvement in the therapeutic activity of liposomal vincristine as measured using a murine P388 lymphocytic leukemia model. At doses above 2 mg/kg, the optimized liposomal vincristine formulation cures greater than 50% of mice bearing the P388 tumor, whereas free vincristine results in no cures.

Animals↗

PubMed1994

Liposome-complement interactions in rat serum: implications for liposome survival studies.

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.

Animals↗

PubMed1994

Influence of plasma on the osmotic sensitivity of large unilamellar vesicles prepared by extrusion.

In the presence of plasma, the osmotic differential required to trigger lysis of large unilamellar vesicles is significantly decreased with the membrane tension at rupture being reduced from about 36 to about 12 dynes/cm for vesicles composed of palmitoyloleoylphosphatidylcholine: cholesterol (55:45). Despite increasing vesicle sensitivity, however, plasma does not alter the characteristics of osmotically induced lysis. As in the absence of plasma, lysis is not an all-or-nothing event but instead results in only partial loss of intravesicular solute, so that following membrane resealing the vesicle interior remains hyperosmotic with respect to the external medium. To identify the component responsible for the observed increase in vesicle osmotic sensitivity, plasma was fractionated by density centrifugation. Albumin and other soluble plasma proteins, including those associated with the complement system, were found to exert only a modest influence on vesicle osmotic behavior. In contrast all of the lipoprotein fractions lowered vesicle tolerance to osmotic pressure, with high density lipoprotein exerting an effect comparable to whole plasma.

Blood Physiological Phenomena↗

PubMed1994

Transfer of liposomal drug carriers from the blood to the peritoneal cavity of normal and ascitic tumor-bearing mice.

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.

Animals↗

PubMed1994

Studies of highly asymmetric mixed-chain diacyl phosphatidylcholines that form mixed-interdigitated gel phases: Fourier transform infrared and 2H NMR spectroscopic studies of hydrocarbon chain conformation and orientational order in the liquid-crystalline state.

Hydrocarbon chain conformational and orientational order in liquid-crystalline bilayers of the highly chain-asymmetric 1-O-eicosanoyl, 2-O-dodecanoyl and 1-O-decanoyl, 2-O-docosanoyl phosphatidylcholines were studied by Fourier transform infrared (FTIR) and deuterium nuclear magnetic resonance (2H-NMR) spectroscopy, respectively, and compared with appropriate symmetric-chain phosphatidylcholines at comparable reduced temperatures. FTIR spectroscopy indicates that these two asymmetric-chain phospholipids contain a slightly greater number of kink, a considerably larger number of double-gauche, but a somewhat smaller number of end-gauche conformers than does dipalmitoylphosphatidylcholine, a symmetric-chain phospholipid having the same total number of carbon atoms in its hydrocarbon chains. Moreover, the asymmetric-chain phospholipids also contain a larger total number of gauche conformers, suggesting that their hydrocarbon chains are more disordered overall than are those of dipalmitoylphosphatidylcholine. 2H-NMR studies of the specifically chain-perdeuterated analogs of these asymmetric-chain lipids reveal that the orientational order parameter profiles of their shorter and longer chains differ both qualitatively and quantitatively, regardless of whether they are esterified at the sn1- or sn2 positions of the glycerol molecule. The longer hydrocarbon chains exhibit unusual orientational order profiles in which the order gradient is steepest in the middle of the chain and relatively shallower in regions adjacent to the carboxyl and methyl termini, whereas the short hydrocarbon chains exhibit orientational order profiles typical of those commonly observed with conventional symmetric chain lipids. When compared at equivalent depths in the bilayer, the shorter hydrocarbon chains of the asymmetric-chain lipids are more orientationally disordered than are their longer chain counterparts. At comparable reduced temperatures, the shorter and longer chains of the asymmetric-chain lipids are more orientationally disordered than those of appropriate short and long symmetric-chain lipids, but the chain-averaged orientational order of the symmetric-chain lipid decreases more sharply with increases in temperature than does that of the comparable chain of the asymmetric-chain species. Moreover, the order plateau regions adjacent to the carboxyl groups of the longer chains of the asymmetric-chain phosphatidylcholines are shorter than those of symmetric-chain lipids of comparable hydrocarbon chain length. Overall, the data indicate that the conformational and orientational order in the liquid-crystalline states of these highly asymmetric-chain lipids differ significantly from those of comparable symmetric-chain lipids. Also, the unusual shape of the orientational order profile of the longer chains of the former is attributed to interaction between the methyl termini regions of the long chains with hydrocarbon chains in opposing monolayers. The latter suggests that some form of hydrocarbon chain interdigitation exists in liquid-crystalline bilayers of these highly asymmetric-chain lipids.

Deuterium↗

PubMed1993

Optimization of the retention properties of vincristine in liposomal systems.

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.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1993

Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients.

The uptake of the anticancer agent doxorubicin into large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (inside acidic) has been investigated using both kinetic and equilibrium approaches. It is shown that doxorubicin uptake into the vesicles proceeds via permeation of the neutral form and that uptake of the drug into LUVs with an acidic interior is associated with high activation energies (Ea) which are markedly sensitive to lipid composition. Doxorubicin uptake into egg-yolk phosphatidylcholine (EPC) LUVs exhibited an activation energy of 28 kcal/mol, whereas for uptake into EPC/cholesterol (55:45, mol/mol) LUVs Ea = 38 kcal/mol. The equilibrium uptake results obtained are analyzed in terms of a model which includes the buffering capacity of the interior medium and the effects of drug partitioning into the interior monolayer. From the equilibrium uptake behaviour, a doxorubicin partition coefficient of 70 can be estimated for EPC/cholesterol bilayers. For a 100 nm diameter LUV, this indicates that more than 95% of encapsulated doxorubicin is partitioned into the inner monolayer, presumably located at the lipid/water interface. This is consistent with 13C-NMR behaviour as a large proportion of the drug appears membrane associated after accumulation as reflected by a broadening beyond detection of the 13C-NMR spectrum. The equilibrium accumulation behaviour of a variety of other lipophilic amines is also examined in terms of the partitioning model.

Amines↗

PubMed1993

The presence of GM1 in liposomes with entrapped doxorubicin does not prevent RES blockade.

The incorporation of ganglioside GM1 or phosphatidylethanolamine-polyethyleneglycol conjugates into liposomes can result in extended circulation lifetimes in vivo. This has been attributed to an ability to avoid uptake by the reticuloendothelial system (RES), specifically the phagocytic cells of the liver and spleen. Here we examine whether a representative large unilamellar vesicle (LUV) formulation which contains GM1 (distearoylphosphatidylcholine/cholesterol/GM1, 45:45:10 mol/mol), actually does avoid the RES. It is shown that a pre-dose of LUVs which contain GM1 and entrapped doxorubicin blocks the accumulation of subsequently injected empty distearoylphosphatidylcholine/cholesterol liposomes in liver. It is therefore concluded that liposomes exhibiting extended circulation lifetimes can induce RES blockade and do not avoid uptake by liver phagocytes.

Animals↗