Endocytosis of liposomes by Entamoeba histolytica and transport of encapsulated molecules toward the cytoplasm

We have examined the interaction of a highly phagocytosing cell: Entamoeba histolytica with liposomes of different lipid compositions, and followed, by a semi-quantitative method, the intracellular fate of the entrapped molecules. Liposomes containing a small molecule, 6-carboxyfluorescein, are first phagocytosed. Then the encapsulated compound migrates from the vacuoles to the cytoplasm. Liposomes containing macromolecular substances, such as fluorescent albumin or ferritin, are also phagocytosed, but the encapsulated molecules remain within the vacuoles. We conclude that the transfer of carboxyfluorescein does not involve a fusion between liposomes and vacuoles, but more likely occurs via diffusion through membranes. The lipid composition of the liposomes does not affect phagocytosis of liposomes. In contrast, oleic acid greatly increases the transfer of carboxyfluorescein from vacuole to cytoplasm.     

Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents

Ligand conjugation is an attractive approach to rationally modify the poor pharmacokinetic behavior and cellular uptake properties of antisense oligonucleotides. Polyethylene glycol (PEG) attachment is a method to increase solubility of oligonucleotides and prevent the rapid elimination, thus increasing tissue distribution. On the other hand, the attachment of long PEG chains negatively influences the pharmacodynamic effect by reducing the hybridization efficiency. We examined the use of short PEG ligands on the in vitro effect of antisense agents. Circular dichroism showed that the tethering of PEG₁₂-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand. In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate.     

Microtubule arrays in fucoid zygotes are sensitive to cytoplasmic pH

Regulation of microtubule (MT) arrays and embryo‐genesis by cytoplasmic pH (pH_c) was investigated in zygotes of the brown alga Pelvetia compressa (J. Agardh) De Toni. pH_c was clamped to (set to) acidic values using a weak acid, propionic acid (PA), and to alkaline values using a weak base, methylamine (MA). Acidification of pH_c from the normal value of 7.4–7.5 to about 7.0 caused disruption of microtubule arrays. The nucleating activity was delocalized from the centrosomes and dispersed over the nuclear envelope, the number of MTs decreased, and MTs failed to extend into the cell cortex. Alkalinization to about pH 8.0 also caused dispersal of nucleating activity, but distinct centrosomes remained. MTs coursed in various directions following MA treatment, giving the array a disorganized appearance. Two MT‐dependent processes, rhizoid morphogenesis and cell division, were found to be perturbed by small changes in pH_c.     

Essential oils encapsulated in liposomes: a review

Abstract

In the recent years there has been an increased interest toward the biological activities of essential oils. However, essential oils are unstable and susceptible to degradation in the presence of oxygen, light and temperature. So, attempts have been made to preserve them through encapsulation in various colloidal systems such as microcapsules, microspheres, nanoemulsions and liposomes. This review focuses specifically on encapsulation of essential oils into liposomes. First, we present the techniques used to prepare liposomes encapsulating essential oils. The effects of essential oils and other factors on liposome characteristics such as size, encapsulation efficiency and thermal behavior of lipid bilayers are then discussed. The composition of lipid vesicles membrane, especially the type of phospholipids, cholesterol content, the molar ratio of essential oils to lipids, the preparation method and the kind of essential oil may affect the liposome size and the encapsulation efficiency. Several essential oils can decrease the size of liposomes, homogenize the liposomal dispersions, increase the fluidity and reduce the oxidation of the lipid bilayer. Moreover, liposomes can protect the fluidity of essential oils and are stable at 4–5?°C for 6 months at least. The applications of liposomes incorporating essential oils are also summarized in this review. Liposomes encapsulating essential oils are promising agents that can be used to increase the anti-microbial activity of the essential oils, to study the effect of essential oils on cell membranes, and to provide alternative therapeutic agents to treat several diseases.     

Oxidation and denaturation of hemoglobin encapsulated in liposomes

A study was made of the in vitro stability of hemoglobin-containing liposomes (‘hemosomes’) prepared from phosphatidylcholines, equimolar cholesterol and red cell lysate by the hand-shaking and ether-injection methods. Absorption spectra indicated hemichrome formation in ‘hemosomes’ prepared by the ether-injection technique, and increased oxidation of hemoglobin in hand-shaken ‘hemosomes’. The denaturation of hemoglobin in ether-injection ‘hemosomes’ was increased if the initial methemoglobin content of the hemolysate, or the temperature of preparation was elevated. It was slower if liposomes were prepared under either N₂ or CO, or if the radical scavenger 1,3-diphenylisobenzofuran was added with the ether. Egg phosphatidylcholine and synthetic saturated phospholipids gave the same results. With hand-shaken ‘hemosomes’ the oxidized product was primarily methemoglobin, and oxidation could be inhibited by using saturated phosphatidylcholines instead of egg phosphatidylcholine. Lysophosphatidylcholine levels were higher and arachidonic acid levels lower in egg phosphatidylcholine ‘hemosomes’ than in equivalent liposomes containing no hemolysate. The ‘hemosome’ seems to be a suitable model for the study of hemoglobin-lipid membrane interactions and the resulting hemoglobin denaturation process.     

Prolonged cardioprotective effect of pyridostigmine encapsulated in liposomes

AimsThe purpose of the present work was to investigate the ability of pyridostigmine encapsulated in long-circulating liposomes, to protect against ECG (electrocardiogram) alterations induced by sympathetic stimulation in rats.Main methodsThe encapsulation of pyridostigmine was carried out by freeze–thaw and extrusion. Blood pressure and ECG (limb lead II) were monitored in anaesthetized male Wistar rats. The formulation containing pyridostigmine was intravenously administrated in 0.1, 0.3 and 1.0 mg/kg doses, and sympathetic stimulation was conducted by administration of 1 or 3 μg of noradrenaline (NA) after 1, 2, 4 or 6 h. The obtained cardiovascular parameters were compared to animals that received intravenous injection of pyridostigmine in free form or saline.Key findingsAfter saline, NA induced a significant increase in QT interval (22.3% after 3.0 μg). Previous administration of free pyridostigmine significantly prevented the increase of QT interval after sympathetic stimulation and the most prominent effect was observed after 1 h for the dose of 0.3 mg/kg (6.8% after 3.0 μg of NA) and was no longer observed after 2 h of the treatment. On the other hand, the maximum effect of pyridostigmine in liposomal formulation preventing QT interval increase was observed 2 h after treatment (9.7% after 3.0 μg of NA) and was still present until 6 h when 1 mg/kg was previous administrated.SignificanceThe results of the present study, beyond to confirm the cardioprotective action of pyridostigmine, suggest that liposomal pyridostigmine may be a potential therapeutic alternative to prevent cardiovascular disturbances resulting from sympathetic hyperactivity.     

pH-sensitive liposomes are efficient carriers for endoplasmic reticulum-targeted drugs in mouse melanoma cells

Tyrosinase, the key enzyme of melanin biosynthesis, is inactivated in melanoma cells following the incubation with the imino-sugar N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum N-glycosylation processing. We have previously shown that tyrosinase inhibition requires high NB-DNJ concentrations, suggesting an inefficient cellular uptake of the drug. Here we show that the use of pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate for the delivery of NB-DNJ reduced the required dose for tyrosinase inhibition by a factor of 1000. The results indicate that these pH-sensitive liposomes are efficient carriers for imino-sugars delivery in the endoplasmic reticulum of mammalian cells.     

Store-operated Ca2+-channels are sensitive to changes in extracellular pH

The sensitivity of store-operated Ca(2+)-entry to changes in the extra- and intracellular pH (pH(o) and pH(i), respectively) was investigated in SH-SY5Y human neuroblastoma cells. The intracellular Ca(2+)-stores were depleted either with 1 mM carbachol (CCH) or with 2 microM thapsigargin (TG). Extracellular acidification suppressed both the CCH- and TG-mediated Ca(2+)-entry while external alkalinization augmented both the CCH- and the TG-induced Ca(2+)-influx. Mn(2+)-quenching experiments revealed that the rates of Ca(2+)-entry at the thapsigargin- or carbachol-induced plateau were both accelerated at pH(o) 8.2 and slowed down at pH(o) 6.8 with respect to the control at pH(o) 7.4. Alteration of pH(o) between 6.8 and 8.2 did not have any significant prompt effect on pH(i) and changes in pH(i) left the CCH-induced Ca(2+)-entry unaffected. These findings demonstrate that physiologically relevant changes in pH(o) affect the store-operated Ca(2+)-entry in SH-SY5Y cells and suggest that endogenous pH(o) shifts may regulate cell activity in situ via modulating the store-operated Ca(2+)-entry.     

Proteinases encapsulated in stimulated release liposomes for cheese ripening

Chymosin, a neutral proteinase from Bacillus subtilis and cardoon cyprosins, were co-encapsulated with phospholipase C in stimulated release liposomes. Encapsulated enzymes were added separately to milk to make cheese. Chymosin and the neutral proteinase accelerated as-casein degradation in comparison with control cheese, whereas b-casein degradation was accelerated by neutral proteinase and cyprosins. Neutral proteinase yielded the highest increase in soluble nitrogen. Cheese flavour intensity was enhanced by the neutral proteinase and cyprosins but not by chymosin.     

Cytogenetic effects of sodium azide encapsulated in liposomes on heteroploid cell cultures.

Lipid vesicles (liposomes) have been shown to be a useful vehicle for the delivery of a variety of compounds to cultured cells. Using multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) we were able to deliver the classical mutagen, sodium azide, into human heteroploid HEp-2 cells. With this method sodium azide is not diluted in culture medium, but it is 'focused' into cells, producing chromosomal aberrations and other major genetic damages. Our results indicate that liposomes are suitable vectors for introducing clastogenic substances into cultured human cells.     

Evidence for H-2-linked control of retrovirus production in Friend virus-induced tumor cell lines.

In Friend leukemia virus-induced tumor cell lines derived from mice congenic with respect to the H-2 complex, most cell lines expressing the H-2k haplotype continuously produced infectious exogenous virus in culture, whereas most cell lines expressing the H-2b or H-2d haplotype stopped producing virus during in vitro passage. This apparent H-2-linked control of virus production did not appear to be the result of alteration of the provirus or resistance to superinfection. The implications of this finding with respect to virus-induced leukemogenesis are discussed.     

Induction of chromosomal aberrations by restriction endonucleases encapsulated in liposomes

We used liposomes to deliver the restriction endonucleases BamHI and SmaI into human heteroploid HEp-2 cells. With this method very low concentrations of enzymes (2 units/ml) were active in the production of chromosomal aberrations. SmaI, which produces blunt-ended double-strand breaks in the DNA molecule, induces chromosomal aberrations more effectively than BamHI, which produces cohesive ends. Our results indicate that liposomes are suitable vectors for introducing restriction endonucleases into cultured human cells.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

Treatment of experimental visceral leishmaniasis with lymphokine encapsulated in liposomes

Highly susceptible mice were infected with Leishmania donovani chagasi and were treated with supernatants, free or encapsulated in liposomes, from concanavalin A-stimulated or unstimulated mouse spleen cell cultures. Treatment consisted of multiple i.v. injections beginning 2 days before to 2 days after infection. Mice treated with lymphokine-rich supernatants encapsulated in liposomes had significantly fewer liver parasites than the control groups, demonstrating in vivo activity of lymphokine against an infectious organism.     

Genetic transformation of yeast protoplasts with DNA encapsulated in liposomes

Abstract Protoplasts of auxotrophic strains of Saccharomyces cerevisiae could be transformed to prototrophy with plasmid DNA encapsulated in liposomes. With negatively charged liposomes, the transformation efficiency was higher than with naked DNA.     

Chromosome mapping of Rfv3, a host resistance gene to Friend murine retrovirus.

Inoculation of adult mice with Friend virus complex usually induces rapid viremia and erythroleukemia, resulting in death in 1 to 3 months. In certain mouse strains, a single host gene, Rfv3, controls the ability to mount a virus-specific neutralizing antibody response which results in elimination of viremia. In this study, microsatellite markers were used to localize the Rfv3 gene to a 20-centimorgan region of mouse chromosome 15 unlinked to immunoglobulin loci, T-cell receptor loci, or the major histocompatibility complex. Potential candidate genes for Rfv3 are several genes expressed in cells of the immune system and previously mapped to the same region, including a T-cell antigen gene, Ly6, and three cytokine receptor genes, IL2rb, IL3rb1, and IL3rb2.     

Determination of transmembrane pH gradients and membrane potentials in liposomes.

Techniques for determining large transbilayer pH gradients (delta pH) and membrane potentials (delta psi) induced in response to delta pH in large unilamellar vesicle liposomal systems by measuring the transbilayer redistribution of radiolabeled compounds have been examined. For liposomes with acidic interiors, it is shown that protocols using radiolabeled methylamine in conjunction with gel filtration procedures to remove untrapped methylamine provide accurate measures of delta pH in most situations. Exceptions include gel state lipid systems, where transbilayer equilibration processes are slow, and situations where the interior buffering capacity is limited. These problems can be circumvented by incubation at elevated temperatures and by using probes with higher specific activities, respectively. Determination of delta pH in vesicles with a basic interior using weak acid probes such as radiolabeled acetate in conjunction with gel filtration was found to be less reliable, and an alternative equilibrium centrifugation protocol is described. In the case of determinations of the membrane potentials induced in response to these pH gradients, probes such as tetraphenylphosphonium and thiocyanate provide relatively accurate measures of the delta psi induced. It is shown that the maximum transmembrane pH gradient that can be stably maintained by an egg phosphatidylcholine-cholesterol 100-nm-diam large unilamellar vesicle is approximately 3.7 units, corresponding to an induced delta psi of 220 mV or transbilayer electrical field of 5 x 10(5) V/cm.     

Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids.

Multilameller liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside GM1, monosialoganglioside GM2, monosialoganglioside GM3, disialoganglioside GD1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distributions of 14C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome preparation. Liver uptake up encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides, regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing GD1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing GD1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.