Jean R. Philippot

A very mild method allowing the encapsulation of very high amounts of macromolecules into very large (1000 nm) unilamellar liposomes

Abstract Using detergent removal by dialysis we have succeeded in making liposomes under very mild conditions, compatible with the introduction of macromolecules such as antibodies or RNA. We observed that in the presence of detergents, Bio-Beads SM-2® adsorb both octylglucoside and Triton X-100 as well as phospholipids. We used octylglucoside, which can be removed by dialysis more quickly than any other detergent, and can be completely eliminated, unlike the detergents ordinarily used, such as Triton X-100 and sodium deoxycholate. Bio-Beads allowed the complete removal of detergent when placed outside the dialysis bag. Under these conditions, dialysis resulted in the formation of large unilamellar liposomes. Their size depended mainly on the composition of the lipids. The largest liposomes corresponded to a lipid mixture of phosphatidylcholine, phosphatidylserine and cholesterol in a molar ratio of 1:1:1. Entrapment measurements and electron microscopic studies showed that these unilamellar liposomes were very large (1000 nm diameter) and that 45–50% of the macromolecular solutions could be entrapped with 13 mM phospholipids (35 l/mol lipid). These liposomes exhibited all the properties required for the delivery of macromolecules into cultured cells.

PHOSPHOLIPID BIOSYNTHESIS BY PLASMODIUM KNOWLESI INFECTED ERYTHROCYTES: THE INCORPORATION OF PHOSPHOLIPID PRECURSORS AND THE IDENTIFICATION OF PREVIOUSLY UNDETECTED METABOLIC PATHWAYS

Metabolic pathways leading to phospholipid biosynthesis in Plasmodium-infected simian erythrocytes were tested and quantified by incubating leucocyte-free erythrocytes in the presence of labelled precursors. Plasma fatty acids and lysophospholipids both served as sources of the fatty acids required for cellular phospholipid biosynthesis. However, the entry of free fatty acids and lysophospholipids appeared to be controlled by a competitive mechanism. A powerful deacylase-acylase system was detected, the nature and specificity of which remain to be defined. Glycerol-3-phosphate incorporation into cellular lipids accounted for most of the new phospholipid molecules formed in parasitized cells, and into cellular lipids accounted for most of the new phospholipid molecules formed in parasitized cells, and this compound, rather than the lysophospholipids, appeared to be the natural acceptor of the acyl groups. By incorporation of nitrogenous bases into cellular phospholipids, we identified significant pathways not previously detected in Plasmodium-infected erythrocytes: the formation of phosphatidylethanolamine by phosphatidylserine decarboxylation, and the formation of phosphatidylcholine by the methylation of phosphatidylethanolamine. These results, associated with the absence of lipid synthesis in host cells, mean that the enzymes controlling these two pathways could serve as enzymatic markers of parasites.

A new cationic liposome encapsulating genetic material. A potential delivery system for polynucleotides.

The endeavour to enhance gene therapy has led to increased research on the development of simple, efficient and safe delivery systems. This study deals with the use of an artificial cationic lipid on the encapsulation of genetic material in liposomes. The addition of a biologically degradable cationic phospholipid, dipalmitoyl-L-alpha-phosphatidylethanolamine covalently coupled to L-lysine, in a standard liposome formulation allowed us to obtain vesicles with high entrapment of various polynucleotides. Polynucleotide degradation by nucleases is markedly prevented by these liposomes. The preparations were stable in both culture medium and human plasma. This latter finding is consistent with the weak binding of plasma proteins on the liposome surface. The efficiency of this new delivery system was demonstrated in antiviral assays. Finally, these liposomes displayed a relatively low cellular toxicity. All these findings indicate that these cationic vesicles are very suitable for genetic material vehiculation.

Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes: Their use as parasite-specific markers

CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in Plasmodium knowlesi-infected erythrocytes obtained from Macaca fascicularis monkeys. Disrupted infected erythrocytes possess a cholinephosphotransferase activity (1.3 +/- 0.2 nmol phosphatidylcholine/10(7) infected cells per h) 1.5-times higher than the ethanolaminephosphotransferase activity. Optimal activities of both enzymes were observed in the presence of 12 mM MnCl2, which was about 3-times as effective as 40 mM MgCl2 as a cofactor. The two activities had similar dependences on pH and thermal inactivation. Their Arrhenius plots show an identical break at 17 degrees C and the corresponding activation energies below and above the critical temperature were similar for the two activities. Sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, beta-D-octylglucoside and lysophosphatidylcholine strongly inhibited the two activities above their critical micellar concentration, but the first three detergents stimulated the activities at lower concentrations. Saponin (0.004-0.5%) either did not affect the two activities or else increased them. Cholinephosphotransferase and ethanolaminephosphotransferase activities had apparent Km values for the CDP ester of 23.4 and 18.6 microM, respectively. CDPcholine and CDPethanolamine competitively inhibited the ethanolaminephosphotransferase and cholinephosphotransferase activities, respectively. The high selectivity of these activities for individual molecular species of diradylglycerol suggests that substrate specificity is responsible for the various molecular species of Plasmodium-infected erythrocyte phospholipids. However, cholinephosphotransferase and ethanolaminephosphotransferase had different dependences on 1,2-dilauroylglycerol and 1-oleylglycerol, which were substrates for cholinephosphotransferase but not for ethanolaminephosphotransferase under our conditions. These data provide the first characterization of an enzyme involved in the intense lipid metabolism in Plasmodium-infected erythrocytes, and the presence of cholinephosphotransferase demonstrates a biosynthesis of phosphatidylcholine by the Kennedy pathway after infection. Our data suggest that cholinephosphotransferase and ethanolaminephosphotransferase activities could be catalyzed by the same enzyme. Furthermore, since host erythrocytes are devoid of these enzymatic activities, cholinephosphotransferase is a parasite-specific membrane-associated enzyme which can be used as a probe or marker.

Phospholipid metabolism as a new target for malaria chemotherapy. Mechanism of action of D-2-amino-1-butanol

A number of choline and ethanolamine analogs were evaluated as inhibitors of P. falciparum growth in vitro. 1-Aziridineethanol, DL-2-amino-1,3-propranediol and D- or L-2-amino-1-butanol were the most efficient inhibitors of parasite multiplication, with an IC50 of 50-80 microM, whereas numerous other analogs were less active. The effect of D-2-amino-1-butanol on various metabolisms of P. knowlesi-infected simian erythrocytes was studied by incubating these cells with different labeled precursors of phospholipids, nucleic acids, proteins, and with radioactive glucose. In the presence of radioactive glycerol, oleate or lysophosphatidylcholine, the appearance of radioactivity in an unnatural phospholipid indicated that 2-aminobutanol was incorporated into a new PL which accounted for up to 30-40% of the total biosynthesized lipids. This new phospholipid accumulated primarily at the expense of PE biosynthesis and decreased the decarboxylation of phosphatidylserine. These effects were not accompanied, over a large range of concentrations, by any parallel change in nucleic or protein synthesis, nor in glucose metabolism. These data demonstrate that the incorporation of analogs, instead of the natural polar head groups, into cellular phospholipids, and/or modification of phospholipid composition have a deleterious impact on the growth of Plasmodium. It follows that PL metabolism is a crucial process for Plasmodium growth and may constitute a potentially fruitful chemotherapeutic approach to malaria.

Inhibition of the in vitro growth of Plasmodium falciparum by D vitamins and vitamin D-3 derivatives

D vitamins are effective inhibitors of the in vitro intraerythrocytic growth of Plasmodium falciparum. Disappearance of the parasitemia was observed after 48 h contact between infected cells and 5 x 10(-6) M 1 alpha-hydroxycholecalciferol, 5 x 10(-5) M 25-hydroxycholecalciferol (25-OH-D-3), 1 alpha, 25-dihydroxycholecalciferol or 2.5 x 10(-4) vitamin D-2 and D-3. A 48 h pretreatment of healthy erythrocytes with 5 x 10(-5) M 25-OH-D-3 did not change their susceptibility to invasion by the parasite and their ability to support the growth of P. falciparum. Ionomycin, a calcium ionophore, and EGTA prevented parasite development at concentrations greater than 2 x 10(-7) M and 4 x 10(-4) M, respectively, but did not antagonize the inhibitory activity of 25-OH-D-3. Addition of 25-OH-D-3 for 12 or 24 h duration to synchronized cultures, showed that the drug had a schizonticidal action, but was without effect when parasites were in the ring form.

Thiolation of low-density lipoproteins and their interaction with L2C leukemic lymphocytes.

We present here, a new method for coupling sulfhydryl groups (SH) to low-density lipoprotein (LDL) surface. This method uses homocysteine thiolactone (HCTL) which reacts with lysine residues in a very mild manner, and permits the selection of the number of SH bound per LDL. Under our experimental conditions (8 SH/LDL), the affinity of thiolated LDL for the specific receptors and their further internalization by L2C lymphocytes are preserved.

Extemporaneous preparation of large unilamellar liposomes

Direct contact between lipids solubilized by octyl glucoside and Amberlite XAD-2 beads yielded large liposomes (240 nm diameter) with no residual detergent molecules, in less than 10 min. This extemporaneous preparation of liposomes was prepared with a detergent/bead ratio no higher than 0.12 (mumol/mg) and a phosphatidylcholine/phosphatidylserine/cholesterol molar ratio of 1:1:1. The liposomes were mainly unilamellar, as deduced from thin section and freeze-fracture electron micrographs and from measurement of calcein incorporation into the vesicles. The relatively large internal volume of these vesicles (8.9 l/mol lipid) accounts for the high percentage of entrapped material observed. The percentage increased with lipid concentration, but could not be increased above 20% corresponding to 20 mM total lipids.

REEVALUATION, USING MARKER ENZYMES, OF THE ABILITY OF SAPONIN AND AMMONIUM CHLORIDE TO FREE PLASMODIUM FROM INFECTED ERYTHROCYTES*

Saponin and ammonium chloride lysis have been applied for some time to the separation of erythrocyte membranes from malarial-infected erythrocytes, allowing easy isolation of the parasites. We present a reevaluation of the use of saponin and ammonium chloride as tools for isolating Plasmodium (knowlesi or falciparum) parasites. Acetylcholine esterase (EC 3.1.1.7) was used as an erythrocyte membrane marker and CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) as a parasite membrane marker to monitor fractionation by these agents. Both saponin and ammonium chloride produced hemolysis of uninfected and infected erythrocytes, but failed to separate host erythrocyte membrane from the parasite, regardless of its stage. Thus, saponin and ammonium chloride can be used to isolate whole infected erythrocytes, depleted of hemoglobin, by selective disruption of uninfected cells.

The influence of coupling transferrin to liposomes or minibeads on its uptake and fate in leukemic L2C cells

Coupling transferrin to liposomes or minibeads did not affect its uptake by L2C lymphocytes via the Tf specific receptors. The uptake kinetics of Tf conjugated with particles about 50 nm in diameter was as rapid as in the case of native Tf, and the receptors were recycled with a similar turnover time (about 15 min). Contrary to the generally accepted scheme, we found some Tf degradation provoked by cellular uptake. The degradation represented about 10% of the amount of ligand taken up by the cells. It occurred when transferrin was coupled to liposomes, but not when coupled to minibeads.