Single-walled Liposomes Research Articles

Physicochemical and Preclinical Evaluation of Spermine-Derived Surfactant Liposomes for in Vitro and in Vivo siRNA-Delivery to Liver Macrophages.

Herein we report on a liposomal system for siRNA delivery consisting of cholesterol (Chol), distearoylphosphatidylcholine (DSPC), and surfactant TF (1-hydroxy-50-amino-3,4,7,10,13,16,19,22-octaoxa-37,41,45-triaza-pentacontane), a novel spermine derivative (HO-EG8-C12-spermine) which has shown improved siRNA delivery to cells in vitro and in vivo. Predominantly single-walled liposomes with reproducible sizes and moderately broad size distributions were generated with an automated extrusion device. The liposomes remained stable when prepared in the presence of siRNA at N/P ratios of 17-34. However, when mixed with human serum in equal volumes, larger aggregates in the size range of several hundred nanometers were observed by dynamic light scattering. These larger aggregates could potentially limit prolonged in vivo applications. Aggregate formation could be reduced by the addition of a cholesterol-hyperbranched polyglycerol surfactant (hbPG) that sterically shields the liposomal surface against serum induced aggregation. In vitro experiments with murine macrophages utilizing macrophage-specific anti-CD68 siRNA loaded liposomes showed potent and sequence specific reduction of CD68 transcript levels without cytotoxicity. Experiments in mice using intravenous application of CW800 NHS ester labeled liposomes, near-infrared in vivo imaging, and fluorescent assisted cell sorting of inflammatory cells demonstrated an almost quantitative accumulation of these liposomes, with and without hbPG, in the liver and a specific knockdown of CD68 mRNA of up to 70% in liver resident macrophages. It was found that aggregate formation of TF liposomes in serum does not significantly affect in vivo siRNA delivery to these central inflammatory cells of the liver.

Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P min, the bilayer lysis tensions γ l were determined. The increase in P min and γ l with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 °C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.

Liposomal membranes. xiv. fusion of liposomal membranes induced by polyisoprenoids as monitored by fluorescence quenching method

Fusion of the single-walled liposomes of egg phosphatidylcholine as induced by the polyisoprenoids such as solanesol, trans-ethyl decaprenoate (EDP), coenzyme Q 10, and dolichol has been investigated adopting the fluorescence quenching method. Relative efficiency of the polyvisoprenoids employed on the induced fusion of liposomes was a sequence of solanesol ⩽ EDP « CoQ 10, dolichol, which was consistent with the result previously obtained by the dye-release method.

Neutron Small-Angle Scattering from Single-Walled Liposomes of Egg Phosphatidylcholine

Neutron small-angle scattering from single-walled liposomes of egg phosphatidylcholine in D2O solution has been measured in order to determine the geometry of the sonicated small liposomes. The results show that the inner and outer radii or the liposomes are 60 and 100 Å, respectively, and that the statistical deviation or the radius amounts to 20% of the average value of the outer radius.

Structure and synthesis of a lipid-containing bacteriophage. Amphiphilic properties of protein IV of bacteriophage PM2.

Interactions between lipids and the DNA-binding protein (protein IV) purified from bacteriophage PM2 were studied in vitro. The efficiency of incorporation of protein IV into single-walled liposomes was more than 90%. Protein IV embedded in liposomes interacted more strongly with PM2 DNA than protein IV alone. The DNA--protein-IV--liposome complex was relatively stable as observed by sedimentation behavior on a sucrose gradient. The interaction between DNA and the protein-IV--liposome was abolished by tryptic digestion, even though 40% of the protein remained in the vesicle. More than 70% of the amino acids of this embedded peptide segment were hydrophobic. Carboxypeptidase digestion of the protein-IV--liposome caused a release of 20% of the radioactivity of the vesicle without changing the DNA-binding ability of the complexes. Modification of the protein-IV--liposome with the chemical probe, 2,4-dinitrofluorobenzene, and analysis of the tryptic peptides released from the protein-IV--liposome demonstrated that the N-terminal basic amino acid cluster segment responsible for the DNA binding was located on the outer surface of the bilayer. These results support an earlier model in which protein IV anchors itself in the inner leaflet of the PM2 bilayer membrane, interacting with the DNA in the virion.

Energy transfer in artificial membrane systems. Singlet-singlet energy transfer from alloxazines to isoalloxazines in dipamitoyl phosphatidylcholine liposomes and dialkylammonium chloride vesicles

The singlet-singlet energy transfer from alloxazines to isoalloxazines has been investigated in dipalmitoyl phosphatidylcholine (DPPC) liposomes and dioctadecyltrimethylammonium chloride (2C 18NC) vesicles to clarify the role of the artificial membranes in the energy transfer phenomenon. The structures of the artificial membranes were divided into two types: the single-walled (sonicated DPPC) and the multi-compartment vesicles (unsonicated DPPC and sonicated 2C 18NC). In the DPPC single-walled liposomes, the energy of the donor lost by quenching is efficiently transferred to the acceptor via the Förster-type dipole-dipole interaction. In the case of multi-compartment liposomes of DPPC, the mean distance between donor and acceptor is so small because the external surface of a bilayer is in the vicinity of the internal surface of another bilayer. As a consequence, efficiencies both of energy transfer and of energy loss were greater than those in single-walled liposomes. The fluid property of the 2C 18NC bilayer allowed the preferential collisional quenching. The marked reduction in the efficiencies of both energy transfer and energy loss were attributed to the elongation of donor-acceptor distances due to the increase of the size of liposome.

Liposomal membranes. VII. Fusion and aggregation of egg lecithin liposomes as promoted by polysaccharides

Pullulan, one of polysaccharides, induces aggregation of egg lecithin multilamellar liposomes as detected by turbidity of the solution. The turbidity disappears upon treatment with pullulanase, indicating that the aggregation induced by pullulan is reversible. On the other hand, the turbidity induced on single-walled liposomes is not completely reversed by incubation with the enzyme. The phenomenon is interpreted as the involvement of irreversible fusion of liposomes. Thus, the enzyme digestion method provides a novel simple means to distinguish fusion of liposomes from aggregation.

Topological studies of the membrane-binding segment of cytochrome b5 embedded in phosphatidylcholine vesicles.

Carboxypeptidase Y (CPY) digestion of cytochrome b5 incorporated into single-walled liposomes of egg phosphatidylcholine (PC) resulted in the release of 25 amino acid residues form each molecule of the cytochrome and concomitant detachment of the cytochrome from the vesicles. This finding suggests that the COOH-terminus of the incorporated cytochrome is exposed to the outer surface of the liposomal membrane, since the cytochrome-carrying vesicles were shown to be impermeable to macromolecules such as dextran T-40 by isopycnic centrifugation studies. The possibility that CPY had attacked a small amount of free cytochrome b5 which was in equilibrium with the liposome-bound cytochrome could be excluded by the finding that transfer of the bound cytochrome to the unbound state was a much slower process than CPY-induced detachment of cytochrome b5. CPY could also attack the COOH-terminus of cytochrome b5 embedded in dipalmitoyl-PC liposomes even below the phase transition temperature of the synthetic phospholipid. Moreover, the tyrosine residue(s) near the COOH-terminus of the PC vesicle-bound cytochrome could be readily iodinated by the action of lactoperoxidase added externally. These observations indicate that the COOH-terminus of the liposome-bound cytochrome, like its heme-containing, hydrophilic domain, is exposed to the outer surface of the vesicular membrane. The cytochrome detached from the vesicles by CPY digestion could neither rebind to the liposomes nor form an oligomeric aggregate, suggesting that CPY had removed all the amino acid residues which are required for the protein to bind to membranes. The mechanism of CPY-induced detachment of cytochrome b5 from liposomes and the size of the peptide segment which is directly involved in the interaction of the cytochrome with the lipid bilayer membranes are discussed.

Inhibition of the binding of cytochrome b5 to phosphatidylcholine vesicles by cholesterol

The binding of cytochrome b 5 to single-walled liposomes of egg phosphatidylcholine was inhibited by the presence of cholesterol in the lipid bilayer under conditions where a limited amount of liposomes was incubated with the cytochrome. Since similar conditions seem to apply for the binding of cytochrome b 5 to erythrocyte ghosts, this observation supports the conclusion of Enomoto and Sato (Enomoto, K. and Sato, R. (1977) Biochim. Biophys. Acta 466, 136–147) that the localization of cholesterol on the outer surface of the ghost membrane prevents the binding of cytochrome b 5 to this surface. The finding reported by Roseman et al. (Roseman, M.A., Holloway, P.W. and Calabro, M.A. (1978) Biochim. Biophys. Acta 507, 552–556) that cholesterol did not prevent the cytochrome binding to phosphatidylcholine liposomes in the presence of a large excess of liposomes could be confirmed in the present study, but this does not contradict the abovementioned conclusion.

Ca++-induced fusion of proteoliposomes: Dependence on transmembrane osmotic gradient

The fusion of cytochrome oxidase liposomes with liposomes reconstituted with mitochondrial hydrophobic protein is dependent on the presence of an acidic phospholipid in the liposomes and on the addition of Ca++ions. Liposomes which have grown, by fusion, to diameters in excess of 1000 A lose the ability to fuse further, unless an osmotic gradient across the liposome membrane is established, with the internal osmotic pressure higher than the external. At a given Ca++ concentration, the extent to which this second fusion step takes place is determined by the ratio of internal to external osmolarity. Single-walled liposomes with diameters exceeding 1 mumM have been produced by this technique. The data suggest that the thermodynamic driving force for the Ca++-induced fusion is an excess surface free energy which can be supplied by membrane curvature or transmembrane osmotic gradients.