Interaction of positively charged liposomes with erythrocyte membrane. An ultrastructural study

Structural perturbations of positively charged sonicated liposomes (L) induced by incubation with human erythrocytes have been studied by thin-section electron microscopy. It has been found that just after mixing with cells L bind to cell surfaces and fuse with each other, first forming larger L and then flattened multilamellar structures. Successive fusion events occurring in the latter result in a reduced number of bilayers which gradually comes to a single one.     

Polymerization of actin by positively charged liposomes

By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.     

Pleiotropic effects of positively charged liposomes on immune functions

Summary Positively charged liposomes have been shown to inhibit the proliferation of lymphocytes induced by various polyclonal activators. We demonstrated that this inhibition is essentially restricted to early phases of activation. B cell proliferation, induction of suppressor cells, and cytotoxic activities are all profoundly inhibited, whereas T4+ cells response to mitogenic stimulation is only moderately affected. The results are discussed in terms of membrane perturbations potentially induced by liposome-lymphocyte interactions. This work has been supported by grants from the National Sciences and Engineering Council of Canada.     

Actin paracrystalline sheets formed at the surface of positively charged liposomes

Paracrystalline aggregates of F-actin spontaneously assemble at the surface of positively charged liposomes. This single-layered paracrystalline array is made up of parallel and juxtaposed actin filaments aligned in register and showing the typical 36-nm periodicity which corresponds to the half-pitch of the double helix strand. This crystallization of pure actin results from a direct interaction between actin and positively charged lipids and does not occur with negative or neutral lipids.     

Interaction of mitochondrial aspartate aminotransferase with negatively charged lecithin liposomes.

Several kinds of hydrophilic proteins were examined to determine their interaction with artificial liposomes. Mitochondrial aspartate aminotransferase (m-GOT) [EC 2.6.1.1], as well as cytochrome c, was found to interact strongly with negatively charged liposomes. In each case, an appreciable amount of the protein bound to liposomes remained unreleased after raising the salt concentration in the medium. The m-GOT tightly bound to the liposomes was also found to become latent in its enzymatic activity, and could be reversibly activated by solubilization of the liposomes with detergent. This is also the case for cytochrome c, which ceases to be reducible by external reductant, such as dithionite. Furthermore, the tightly bound m-GOT was not susceptible to the proteolytic action of trypsin, or that of Nagarse. From these observations it can be inferred that these basic proteins interact with acidic liposomes not only electrostatically but also hydrophobically. This kind of hydrophobic interaction was not observed in the combination of positively charged liposomes and acidic proteins, including s-GOT. Mitochondrial GOT was shown to be bound to isolated intact mitochondrial, but the bound enzyme was fully active, in contrast to the case of acidic liposomes. The hydrophobic interaction of water-soluble protein with liposomes is discussed in connection with the penetration of matrix enzyme through mitochondrial membranes.     

Deoxynucleic guanidine; synthesis and incorporation of purine nucleosides into positively charged DNG oligonucleotides

The synthesis of purine nucleosides capable of making the guanidinium linkage is described for the first time starting from the corresponding 2'-deoxynucleosides. The positively charged mixed base DNG oligomer containing guanine was synthesized on solid-phase using CPG as support from 3' to 5' direction using the precursor building block nucleosides.     

Interaction of positively charged ubiquinone analog (MitoQ10) with DT-diaphorase from liver mitochondria

Effects of the coenzyme Q analog (MitoQ₁₀) carrying a positively charged decyltetraphenylphosphonium group on functional activity of phosphorylating liver mitochondria were studied. Using inhibitory analysis it was found that at micromolar concentrations this quinone is reduced by NADH-dependent DT-diaphorase. Under conditions of malate oxidation, MitoQ₁₀ stimulates electron transfer from NADH to oxygen by shunting the block of rotenone-induced electron transport in Complex I. Steady-state mitochondrial respiration induced by rotenone and MitoQ₁₀ (1 μM), as well as K3 shunt are both blocked by the DT-diaphorase inhibitor dicumarol, the Complex III inhibitor myxothiazole, and the cytochrome oxidase inhibitor cyanide. The electron transport chain induced in liver mitochondria by MitoQ₁₀ in the presence of rotenone appears as follows: NADH → DT-diaphorase → MitoQ₁₀ → Complex III → Complex IV → O₂. Under conditions of malate (but not succinate) oxidation, MitoQ₁₀ and high concentrations of vitamin K3 induce in mitochondria cyanide-resistant respiration and opening of the nonspecific pore eventually resulting in inhibition of oxidative phosphorylation. It is concluded that MitoQ₁₀ should be regarded as an analog of hydrophilic quinones (vitamin K3, duroquinone, etc.) widely known as substrates for mitochondrial DT-diaphorase not interacting with CoQ₁₀ rather than as a natural CoQ₁₀ analog.     

Encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with fluid liposome-encapsulated antisense oligonucleotides

The catalase-peroxidase encoded by katG of Mycobacterium tuberculosis is a more effective activator of the antibiotic isoniazid than is the equivalent enzyme from Escherichia coli. The environment of the heme iron was investigated using X-ray absorption spectroscopy to determine if differences in this region were associated with the differences in reactivity. The variation in the distal side Fe-ligand distances between the two enzymes was the same within experimental error indicating that it was not the heme iron environment that produced the differences in reactivity. Analysis of variants of the E. coli catalase-peroxidase containing changes in active site residues Arg102 and His106 revealed small differences in Fe-water ligand distance including a shorter distance for the His106Tyr variant. The Arg102Leu variant was 5-coordinate, but His106Cys and Arg102Cys variants showed no changes within experimental error. These results are compared with those reported for other peroxidases.     

Synthesis of positively charged galactosurfactants

An approach to synthesis of cationic carbohydrate surfactants with potential antimicrobial or transfected activities is described.     

Enhanced healing of cutaneous wounds in rats using beads with positively charged surfaces.

The efficacy of electrical fields in soft-tissue repair is unclear. Materials with a charged surface provide a localized charged environment. We examined the effects of surface-charged particles in wound healing in rats with paired dorsal incisions with one side serving as a control. Tensiometry demonstrated that after 10 days, wounds with positively charged particles were 53 percent stronger (p less than 0.001) than controls (10 rats, 30 wound strips), whereas differences with negatively charged (6 rats, 15 strips) or uncharged beads (11 rats, 33 strips) were insignificant. Histologically, wounds with positively charged particles were characterized by large quantities of collagen-rich connective tissue and by prominent bead-associated giant cells. At 94 days, no differences in wound strength were noted. This method of creating charged local environments has potential clinical implications and may add insights into the behavior of cells in response to charged stimuli.     

Effects of oral administration of positively charged insulin liposomes on alloxan diabetic rats: preliminary study.

Insulin encapsulated in liposomes of various lipid compositions were prepared. The amount of insulin trapped in these liposomes increased in the order, negatively charged liposomes less than neutral liposomes less than positively charged liposomes. In positively charged liposomes, the amount of insulin trapped increased with increase in the amount of amphiphile stearylamine. Under the conditions tested, the highest insulin content (about 50%) was obtained with liposomes composed of phosphatidyl choline/cholesterol/stearylamine in a molar ratio of 7/2/2.25. These liposomes were stable on incubation for 3 hr at 37 degrees C in solutions of pepsin, trypsin, and pancreatin, and after these incubations, a considerable amount of insulin was still associated with the liposomes. However, the liposomes released almost all the insulin into the medium on treatment with bile. When the liposomes were administered orally to rats in the 3rd phase of acute alloxan diabetes, reduction of the blood glucose level was observed in 7 of 11 animals, the reduction persisted for several hours and was ranging from 30 to 75%. In alloxan diabetic rats showing hyperglycemia for 3 to 6 months, the liposomes also increased the glucose tolerance in half the animals tested.     

Interaction of human erythrocyte ghosts or liposomes with polyethylene glycol detected by fluorescence polarization

The membrane fluidity of human erythrocyte ghost was temporarily increased by the addition of polyethylene glycol with molecular weight of 7500. On the other hand, the fluidity of dipalmitoylphosphatidyl choline bilayer liposomes was monotonously decreased by the addition of polyethylene glycol. Fusion of liposomes was inhibited by the interaction with polyethylene glycol. The temporary increase in membrane fluidity of erythrocyte ghosts was considered to be the result of the clustering of membrane-bound proteins which is believed to be one of the most important sequences in cell fusion.     

Synthesis of positively charged galactose-bearing surfactants

An approach to the synthesis of cationic carbohydrate surfactants with potential antimicrobial and transfecting activities is proposed.     

Interaction of oligonucleotides with cellular proteins

Oligonucleotides (ODNs) conjugated to 4-[(N-2-chloroethyl-N-methyl)amino] benzylamine were used to investigate ODN-binding proteins in cells of different origin. The data obtained demonstrate that 68, 46, 38 and 28 kDa ODN-binding proteins are universal for tested cell lines.     

Fusion of negatively charged liposomes with clathrin-uncoated vesicles

The interaction of lipid vesicles with uncoated vesicles from bovine brain has been studied by fluorescence energy transfer between fluorescent lipid analogs (NBD-PE, Rh-DOPE), by loss of fluorescence self-quenching (NBD-PE, carboxyfluorescein) and by freeze-fracture electron microscopy. The fluorescence techniques monitor the mixing of membranous lipids and the induced release of encapsulated material. The results demonstrate a mixing of the negatively charged lipid (PA, PS) vesicles with the uncoated vesicles. In parallel with the lipid mixing a release of intravesicularly encapsulated material takes place. Lipid vesicles composed of zwitterionic lipids (PC, DOPC, PC:PE) do not specifically interact with uncoated vesicles. The electron micrographs reveal single fusion events. Studies on the kinetics are consistent with a fusional mechanism of the negatively charged lipid vesicles with uncoated vesicles.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Interaction of ferricytochrome c with liposomes

The binding of ferricytochrome c to liposomes consisting of phosphatidylcholine mixtures with cardiolipin (3:1) or phosphatidylserine (3:1) has been investigated. Experimental data have been analyzed in terms of two-dimensional models of large ligand adsorption. The equilibrium parameters of ferricytochrome c interaction with a phospholipid bilayer are determined.     

Interaction of isopropylthioxanthone with phospholipid liposomes

Isopropylthioxanthone (ITX) is a highly lipophilic molecule which can be released in foods and beverages from the packages, where it is present as photoinitiator of inks in printing processes. Recently it was found in babies milk, and its toxicity cannot be excluded. The structure of the molecule suggests a possible strong interaction with the lipid moiety of biological membranes, and this is the first study of its effects on phospholipid organization, using differential scanning calorimetry (DSC) and spin labelling techniques. The data obtained with multilamellar liposomes of saturated phospholipids of different length, with and without cholesterol, point out that the molecule changes the lipid structure; in particular, in the gel state, behaving like a disordering agent it increases the mobility of the bilayer, while, in the fluid state, tends to rigidify the membrane, in a cholesterol like way. This behavior supports the hypothesis that ITX experiences a relocation process when the lipid matrix passes from the gel to the fluid state.