M. Africa de Madariaga

N-PALMITOYLPHOSPHATIDYLETHANOLAMINE STABILIZES LIPOSOMES IN THE PRESENCE OF HUMAN SERUM : EFFECT OF LIPIDIC COMPOSITION AND SYSTEM CHARACTERIZATION

Liposomes containing negatively-charged phospholipid, N-palmitoylphosphatidylethanolamine (NPPE) were examined for stability in the presence of human serum, using the release of the entrapped 5,6-carboxyfluorescein as an aqueous marker. Either small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV) were used. Incorporation of NPPE into PC SUV decreases leakage in the presence of serum or phosphate-buffered saline, no strictly related to size increase observed and to the surface negative charge present. The stabilizing effect of NPPE and Chol were synergistic. Inhibition of destabilization induced by serum of PC/Chol liposomes was observed when NPPE concentrations were above 12 mol%. Change in the membrane fluidity or incorporation of a monosialoganglioside into liposomes do not significantly change the half-life of liposomes in the presence of a high NPPE concentration. Incorporation of NPPE into PC/Chol liposomes increases membrane rigidity which does not change after serum incubation. The presence of NPPE in liposomes decreases lipid transfer/exchange between liposomes and lipoproteins although the same amount of serum proteins were incorporated as in PC/Chol liposomes. As expected, these proteins are accessible to trypsin digestion. In accordance with these results, the liposome agglutination assay shows no steric barrier activity. As a whole, the results obtained in this paper suggest a complex mechanism for stabilization of NPPE containing liposomes in human serum.

Incorporation of N-acylethanolamine phospholipids into egg phosphatidylcholine vesicles: characterization and permeability properties of the binary systems

We have studied the effect of the N-acylphosphatidylethanolamine (N-acylPE) on the permeability properties of liposomes composed primarily of egg phosphatidylcholine using a fluorescent anionic dye, carboxyfluorescein, as model solute. Leakage from liposomes decreased and vesicle size increased with increasing N-acylPE content. In addition, measurement of the trapped aqueous space, using the same dye marker, showed a correlation between trapped volume and vesicle size determined by dynamic light scattering. Permeability parameters were calculated according to the pseudo-first-order analysis. It appears that N-acylPE stabilizes liposomes at least in part through its ability to impart surface negative charge, in accord with the results obtained with potassium chloride as encapsulated solute. These results agreed well with osmotic response of anionic lipid vesicles. Cholesterol stabilizes N-acylPE liposomes in a proportional manner to the molar fraction of the effector.

Role of headgroup structure in the phase behaviour of N-acylethanolamine phospholipids: hydrogen-bonding ability and headgroup size☆

The physical properties of aqueous dispersions of N-acylphosphatidylethanolamine from natural origin with long N-acyl chain (NAPE) and headgroup modified analogues have been studied. N-Acylation of PE causes a significant increase in the gel-to-liquid crystalline lamellar phase transition temperature in contrast with saturated N-acyl(dipalmitoyl) PEs, and in addition it does not restrict the headgroup rotational mobility in gel phase. The results agree with the increase of hydration of the phosphate group compared with that in PE and suggest the formation of hydrogen bonds between amide groups. The modifications introduced modulate the headgroup size and their hydrogen bonding capability. An increasing number of methylene groups between the phosphate and amide groups does not modify the phase behaviour observed. N-methylation of the amide group, which prevents the possibility of intermolecular hydrogen bond formation, decreases the melting temperature and the cooperativity of the phase transition and does not change the phase behaviour, while the hydration at the ester carbonyl groups level is decreased. On the other hand, the addition of N-ethyl substituent to the amide group or substitution of an ester group for this group increases its tendency to form structures with inverted geometries. The behaviour of these compounds suggests that hydration forces must be more important than considerations of the lipid dynamic shape in predicting the relative stabilities of lamellar vs. non-lamellar phases for NAPEs with long saturated N-acyl chain.

The influence of N-acyl chain length on the phase behaviour of natural and synthetic N-acylethanolamine phospholipids

Abstract The influence of N -acyl chain length on the thermotropic phase behaviour of N -acylphosphatidylethanolamines of natural origin was studied by DSC, 31 P-NMR and IR-FT spectroscopy. The obtained data were also compared with the known behaviour of N -acyl derivates of dipalmitoylphosphatidylethanolamine. All the compounds analyzed form multilamellar liposomes and their enthalpies of calorimetric transition always exceed the corresponding values for phosphatidylcholines (PC) and phosphatidylethanolamines (PE). The transition temperature ( T m ) for both series of compounds shows a V-shaped behaviour with an apparent minimum value at N -C6 and N -C8 chain length for natural and synthetic N -acylamino phospholipids, respectively. This behaviour can be accounted for by the disruptive effect of the terminal methyl group of the N -acyl chain in the bilayer packing. For long N -acyl chain (C18 to C14), they pack in the entire hydrocarbon width of bilayer and the T m is higher than that of the corresponding PE. On decreasing the N -acyl chain length (C12 to C6), the T m value decreases and becomes lower than that of the corresponding PC. The hydrocarbon chain packing in the gel state is perturbed by the N -acyl chain inequivalence and the disordering effect reaches a maximum value. For shorter N -acyl chains (C4 and C2), the T m approaches that of the corresponding PC. This can be related to the smaller disruptive effect due to the location of the methyl terminus in the vicinity of the bilayer interface region. The results obtained by 31 P-NMR and infrared spectroscopy confirm these observations.

Aggregation and fusion of vesicles composed of N-palmitoyl derivatives of membrane phospholipids.

N-Acylphosphatidylethanolamines and N-acylphosphatidylserines have been isolated from mammalian cells and have been associated with some tissue degenerative changes, although the relationship between their synthesis and the uncontrolled sequence of events that ends in irreversible tissue damage is not completely established. Our results show that monovalent and divalent cations induce aggregation and fusion of liposomes constituted by N-palmitoylphosphatidylethanolamine (NPPE) and N-palmitoylphosphatidylserine (NPPS). The effectiveness of cations to induce the aggregation of NPPE and NPPS liposomes is Ca2+>Mg2+>>Na+. NPPS liposomes aggregate at lower concentrations of divalent cations than NPPE liposomes, but with sodium NPPE liposomes aggregate to a higher extent than NPPS liposomes. The reaction order for the aggregation processes depends on the lipid and the cation nature and range from 1.04 to 1.64. Dynamic light scattering shows an irreversible increase of the size of the aggregates in the presence of all cations tested. The irreversibility of the aggregation process and the intermixing of bilayer lipids, as studied by resonance energy transfer assay, suggest that fusion, rather than aggregation, occurs. The existence of a real fusion was demonstrated by the coalescence of the aqueous contents of both NPPS and NPPE liposomes in the presence of either monovalent or divalent cations. The different binding sensitivity of Ca2+ to NPPS and NPPE liposomes, determined by ş potential measurements, agrees with the results obtained in the aggregation and fusion assays. Our results suggest that the synthesis in vivo of N-acylated phospholipids can introduce important changes in membrane-mediated processes.

Cation-induced aggregation and fusion of N-acyl-N-methyl-phosphatidylethanolamine vesicles

Aggregation and fusion of unilamellar vesicles consisting of N-acyl-N-methylphosphatidylethanolamine were studied as a function of mono- and divalent cation concentrations. The aggregation reactions were irreversible processes, as demonstrated by changes in monovalent ion concentrations and by the addition of ethylenediaminetetraacetic acid (EDTA) to chelate divalent cations, suggesting the possibility of some cation-induced vesicle fusion. An increase in the NaCl ionic strength of the vesicle suspension solutions diminishes the threshold concentration for Li+ and K+ and increases that corresponding to Mn2+, Mg2+ and Ca2+. However NaCl concentrations above 300 mM yield smaller threshold values for the divalent cation-induced processes, probably due to the increased size of phospholipid vesicles as the ionic strength of the medium increases.

Fourier transform infrared spectroscopic analysis of the ester and amide bands of the hydrated dispersions of N-acylethanolamine phospholipids

Abstract Fourier transform infrared spectroscopy was used to characterize the stretching vibrations of the ester carbonyl groups and the amide I vibration of the amide group of N-acylphosphatidylethanolamine (N-acylPE) bilayers. To obtain quantitative information about changes in hydration, band profiles were simulated with Gaussian-Lorentzian functions. The results are particularly significant since they probably reflect different changes in the hydrogen-bonding interactions in the polar/apolar interfacial region at the gel-liquid-crystalline phase transition of the N-acylPE bilayers related to the N-acyl chain length. In the same way, the carbonyl ester interfacial region of N-acylPEs is spectroscopically similar to those of phosphatidylethanolamine bilayers, in which two populations of hydrogenbonded ester carbonyl groups were observed. Moreover, our study provides additional data pertinent to the physical basis of the thermotropic phase behaviour of aqueous dispersions of N-acylPEs and the basis of the differences between these bilayers and the more intensively studied phosphatidylcholine and phosphatidylethanolamine bilayers.

Photodynamic inactivation of viruses by immobilized chlorin- containing liposomes

The viral safety of blood derived products relies in properly chosen inactivation procedures. In this way, it has been reported that some photosensitizers are useful products for blood sterilization. The data presented here show the high incorporation efficiency of the chlorin 3-phorbinepropanol, 9,14-diethyl-4,8,13,18-tetramethyl-20-(3S-trans) (CHL) into anionic unilamellar liposomes, give a protocol for the steric immobilization of chlorin-containing liposomes in a chromatographic support and provide the studies of photodynamic inactivation of bovine viral diarrhea virus (BVDV) and encephalomyocarditis virus (EMCV) with chlorin-containing liposomes, free in solution and immobilized on Sephacryl S-1000 beads. The study demonstrates the successful inactivation of the enveloped virus BVDV by both preparations in culture medium and the resistance of the non-enveloped virus EMCV. The effectiveness of CHL-containing liposomes, in solution and immobilized in the chromatographic support, decreased when the culture media was replaced with human blood plasma. Moreover, the reduction factor of the virus titer after irradiation was smallest when immobilized liposomes were used. Nevertheless, the reduction factor for the virus titers of enveloped viruses after irradiation of human blood plasma samples with immobilized chlorin-containing liposomes increased with the reduction of the sample thickness. The more outstanding aspect of this paper is the design of a system useful for blood sterilization that can be easily removed after photodynamic treatment and, therefore, able to be applied in the manufacturing processes.

N-Stearoyl-phosphatidylserine: Synthesis and role in divalent-cation-induced aggregation and fusion

N-Acylphosphatidylserines have been isolated from intact and injured tissues, but the participation of such acidic phospholipids in membrane aggregation and fusion has not been demonstrated. We have synthesized N-stearoylphosphatidylserine (NSPS) and examined divalent-cation-induced aggregation of NSPS-liposomes, which leads to membrane destabilization and fusion. The purified lipid was characterized by its chromatographic and spectroscopic (infrared and 1H nuclear magnetic resonance) properties and by its chemical degradation pattern. Aggregation of unilamellar NSPS-liposomes was studied as a function of calcium and magnesium concentration. The ability of calcium and magnesium to induce vesicle aggregation is higher for phosphatidylserine (PS)-liposomes (threshold concentration 1.5 mM for calcium and 4.6 mM for magnesium) than for NSPS-liposomes (threshold concentration 2.8 mM for calcium and 6.6 mM for magnesium). The irreversibility of the aggregation reactions after adding EDTA suggests that vesicle fusion might occur in the presence of calcium and magnesium. Preliminary studies, based on mixing of both lipid and internal aqueous contents, show that fusion rather than aggregation of NSPS-liposomes occurs in the presence of calcium ions. The tendency of NSPS-liposomes to aggregate at higher cation concentrations than PS-liposomes suggests that N-acylation of phosphatidylserine protects the membrane against degenerative damage caused by aggregation and fusion.

Molecular organization of hydrated dispersions of N-acylethanolamine phospholipids and mixtures with phosphatidylcholine

Abstract The physical properties of aqueous dispersions of N-acylphosphatidylethanolamine (N-acylPE) of natural origin with a long N-acyl chain have been studied and compared with those of phosphatidylcholine (PC). Saturated N-acylPEs form lamellar structures with a restricted head-group rotational mobility and with the same motional freedom in the hydrophobic area as PC. However, the presence of unsaturations in N-acylPE increases the mobility of the head group in the gel phase compared to that of the synthetic saturated analogue. The presence of an additional hydrocarbon chain and the suggested formation of hydrogen bonds between amide groups produces a significant increase in the thermodynamic parameters (transition temperature and enthalpy) of the main calorimetric transition. Another stabilizing factor could be related to the increase in the degree of hydration of the interfacial and head-group molecular regions of N-acylPE with respect to those of PC. Analysis of mixtures of N-acylPE and PC reveals that both phospholipids were miscible over the entire range of composition assayed. When increasing the PC content of N-acylPE bilayers, decreases in the transition temperature, transition enthalpy and cooperativity of the main transition occur. Although the mixing of the components is clearly nonideal, no lateral phase separation of the components occurs in the bilayer of the binary system. The polymorphic behaviour of the binary systems observed by 31P-NMR reveals a hydrated lamellar phase spectrum composite of the two spectral line shapes corresponding to both phospholipids with the same chemical shift anisotropy of the monocomponent systems.