Margarita Mora

Kinetics of singlet oxygen photosensitization in human skin fibroblasts

The roles played by singlet oxygen ((1)O(2)) in photodynamic therapy are not fully understood yet. In particular, the mobility of (1)O(2) within cells has been a subject of debate for the last two decades. In this work, we report on the kinetics of (1)O(2) formation, diffusion, and decay in human skin fibroblasts. (1)O(2) has been photosensitized by two water-soluble porphyrins targeting different subcellular organelles, namely the nucleus and lysosomes, respectively. By recording the time-resolved near-IR phosphorescence of (1)O(2) and that of its precursor the photosensitizers triplet state, we find that the kinetics of singlet oxygen formation and decay are strongly dependent on the site of generation. (1)O(2) photosensitized in the nucleus is able to escape out of the cells while (1)O(2) photosensitized in the lysosomes is not. Despite showing a lifetime in the microsecond time domain, (1)O(2) decay is largely governed by interactions with the biomolecules within the organelle where it is produced. This observation may reconcile earlier views that singlet oxygen-induced photodamage is highly localized, while its lifetime is long enough to diffuse over long distances within the cells.

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.

Do folate-receptor targeted liposomal photosensitizers enhance photodynamic therapy selectivity?

One of the current goals in photodynamic therapy research is to enhance the selective targeting of tumor cells in order to minimize the risk and the extension of unwanted side-effects caused by normal cell damage. Special attention is given to receptor mediated delivery systems, in particular, to those targeted to folate receptor. Incorporation of a model photosensitizer (ZnTPP) into a folate-targeted liposomal formulation has been shown to lead an uptake by HeLa cells (folate receptor positive cells) 2-fold higher than the non-targeted formulation. As a result, the photocytotoxicity induced by folate-targeted liposomes was improved. This selectivity was completely inhibited with an excess of folic acid present in the cell culture media. Moreover, A549 cells (folate receptor deficient cells) have not shown variations in the liposomal incorporation. Nevertheless, the differences observed were slighter than expected. Both folate-targeted and non-targeted liposomes localize in acidic lysosomes, which confirms that the non-specific adsorptive pathway is also involved. These results are consistent with the singlet oxygen kinetics measured in living cells treated with both liposomal formulations.

Design and characterization of liposomes containing long-chain N-acylPEs for brain delivery: penetration of liposomes incorporating GM1 into the rat brain.

AbstractPurpose. To develop a suitable liposomal carrier to encapsulate neu- roactive compounds that are stable enough to carry them to the brain across the blood-brain barrier with the appropriate surface characteristics for an effective targeting and for an active membrane transport. Methods. Liposomes containing glycosides and a fusogenic lipid were prepared by extrusion. Photon correlation spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry were used to characterize liposomal preparations. Tissue distribution was determined by using 3H-cholesterylhexadecylether as a marker. Results. The incorporation of glycoside determinants and N-palmitoylphosphatidylethanolamine gives liposomes with similar initial size, trapped volume, negative surface charge, bilayer fluidity, and melting temperature, except for monosialoganglioside-containing liposomes, which showed less negative surface charge and the highest size, trapped volume and melting temperature. All glycosilated formulations gave liposomes able to retain up to the 95% of encapsulated carboxyfluorescein after 90 min at physiologic temperature even in the presence of serum. Monosialoganglioside liposomes were recovered in the cortex, basal ganglia, and mesencephalon of both brain hemispheres. The liver uptake was higher for sulfatide- and glucose-liposomes, whereas the higher blood levels were observed for glucose- and mannose-liposomes. Conclusions. These results show the suitability of such liposomal formulations to hold encapsulated drugs. Moreover, the brain uptake of monosialoganglioside liposomes makes them good candidates as drug delivery systems to the brain.

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.

Cellular and vascular effects of the photodynamic agent temocene are modulated by the delivery vehicle

The effects of the drug delivery system on the PDT activity, localization, and tumor accumulation of the novel photosensitizer temocene (the porphycene analogue of temoporfin or m-tetrahydroxyphenyl chlorin) were investigated against the P815 tumor, both in vitro and in DBA/2 tumor bearing mice. Temocene was administered either free (dissolved in PEG(400)/EtOH mixture), or encapsulated in Cremophor EL micelles or in DPPC/DMPG liposomes, chosen as model delivery vehicles. The maximum cell accumulation and photodynamic activity in vitro was achieved with the free photosensitizer, while temocene in Cremophor micelles hardly entered the cells. Notwithstanding, the micellar formulation showed the best in vivo response when used in a vascular regimen (short drug light interval), whereas liposomes were found to be an efficient drug delivery system for a tumor cell targeting strategy (long drug-light interval). PEG/EtOH formulation was discarded for further in vivo experiments as it provoked lethal toxic effects caused by photosensitizer aggregation. These results demonstrate that drug delivery systems modulate the vascular and cellular outcomes of photodynamic treatments with temocene.

Molecular and physicochemical aspects of the interactions of the tuberculostatics ofloxacin and rifampicin with liposomal bilayers: a 31P-NMR and DSC study

Abstract ζ-Potential measurements were obtained in order to determine whether electrostatic interactions occur between the tuberculostatics ofloxacin and rifampicin and the molecular species of different lipid bilayers. The results obtained showed that both ofloxacin and rifampicin modify the surface charge of lipid vesicles. The extent of these modifications depends on the nature of both the bilayer constituents and the tuberculostatics: a relationship was found between the initial charge at the bilayer surface and the ζ-potential measured changes and so, no significant changes were observed when the presence of anionic lipids in the bilayer was lower than 10%. The highest percentage of variation was for N-palmitoylphosphatidylethanolamine (NPPE)-containing liposomes, which have the highest negative charge. 31P-NMR experiments showed the ability to adopt the bilayer structure of all the mixtures assayed, although changes in the shape of the resulting spectra were observed in function of both the nature of the molecular constituents of the bilayer and the temperature. The nature of DPPC and DSPC gave rigid bilayers, when the temperature was below that of their transition, whereas the incorporation of NPPE allowed fluid structures above and below the transition temperature of the NPPE-containing mixtures to be obtained. In the presence of the tuberculostatic drugs ofloxacin and rifampicin no fundamental structural changes, e.g. the formation of hexagonal arrangements were observed in the structure of the membrane. However, 31P-NMR spectra of NPPE-containing liposomes underwent a decrease in their intrinsic line width, though no significant changes were observed in the chemical shift anisotropy, when ofloxacin and rifampicin were added to the lipid mixtures at a 1:10 weight ratio. This result was supported by the considerable degree of liposome–drug interactions observed by ζ-potential measurements when liposomes contained NPPE. The DSC study of the effects of ofloxacin and rifampicin on the thermotropic behaviour of the different lipid bilayers showed no significant changes in the main transition temperature of the mixture after the incorporation of these tuberculostatics. The ΔHcal and the ΔScal values obtained from the calorimetric thermograms were dependent on the lipid composition, were slightly modified in the presence of ofloxacin and fell after the incorporation of rifampicin to the bilayers. The degree of cooperativity only showed appreciable changes when ofloxacin was incorporated into NPPE- and GM1-containing liposomes. ζ-Potential measurements, 31P-NMR experiments and DSC thermotropic studies prove that it is possible to obtain stable preparations of liposomes containing ofloxacin and rifampicin for use in tuberculosis therapy.

Temocene: the porphycene analogue of temoporfin (Foscan®)

Temocene, a porphycene analogue of temoporfin, has been synthesized. Compared to temoporfin, temocene is endowed with 2.5-fold larger absorption coefficients in the red part of the spectrum while keeping its excellent photophysical and singlet oxygen photosensitization ability. While its photodynamic activity towards HeLa cells is lower than that of temoporfin, its higher photostability, lower dark toxicity and mitochondrial localisation make temocene a promising candidate for photodynamic therapy applications.