Maria Lluïsa Sagristá

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.

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

BACKGROUND The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments. METHODS Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence. RESULTS m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments. CONCLUSIONS Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism. GENERAL SIGNIFICANCE These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.

Poly(D, L-lactide-co-glycolide) nanoparticles as delivery agents for photodynamic therapy: enhancing singlet oxygen release and photototoxicity by surface PEG coating.

Poly(D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) are being considered as nanodelivery systems for photodynamic therapy. The physico-chemical and biological aspects of their use remain largely unknown. Herein we report the results of a study of PLGA NPs for the delivery of the model hydrophobic photosensitizer ZnTPP to HeLa cells. ZnTPP was encapsulated in PLGA with high efficiency and the NPs showed negative zeta potentials and diameters close to 110 nm. Poly(ethylene glycol) (PEG) coating, introduced to prevent opsonization and clearance by macrophages, decreased the size and zeta potential of the NPs by roughly a factor of two and improved their stability in the presence of serum proteins. Photophysical studies revealed two and three populations of ZnTPP and singlet oxygen in uncoated and PEGylated NPs, respectively. Singlet oxygen is confined within the NPs in bare PLGA while it is more easily released into the external medium after PEG coating, which contributes to a higher photocytotoxicity towards HeLa cells in vitro. PLGA NPs are internalized by endocytosis, deliver their cargo to lysosomes and induce cell death by apoptosis upon exposure to light. In conclusion, PLGA NPs coated with PEG show high potential as delivery systems for photodynamic applications.

Anthracene-based fluorescent nanoprobes for singlet oxygen detection in biological media.

We have developed a novel singlet oxygen nanoprobe based on 9,10-anthracenedipropionic acid covalently bound to mesoporous silica nanoparticles. The nanoparticle protects the probe from interactions with proteins, which detract from its ability to detect singlet oxygen. In vitro studies show that the nanoprobe is internalized by cells and is distributed throughout the cytoplasm, thus being capable of detecting intracellularly-generated singlet oxygen.

Effect of germinated seeds extract on the respiratory activity of human skin fibroblasts and sheep liver mitochondria. Influence on cell viability and proliferation and their uselfulness as active cosmetic ingredient

The present work studies the ability of a germinated seeds extract (alfalfa, radish, wheat, soy) to influence cell respiration of skin fibroblasts and the respiratory activity of sheep liver isolated mitochondria, and the cytotoxicity of the product on cultured skin fibroblasts. The content of free and total amino acids present in this extract was determined. Aspartic acid represented 37.52% of free amino acids in the extract, but 48.07% glutamic acid was found when total amino acids obtained by acid hydrolysis were quantified. The extract contains more than 6.5% free serine, glutamic acid and alanine. Arginine, serine, aspartic acid and threonine are the other amino acids that represent more than 4.5% total amino acids. Respiration of sheep liver mitochondria and human skin fibroblasts (Foreskin ATCC CRL‐1635) was checked polarographically. The results obtained show the ability of this extract to stimulate cellular respiration of both in vitro models, but the effect is more marked on cell cultured fibroblasts, and its dose dependency. The role of acidic amino acids, aspartic and glutamic acids, on the increase of oxygen consumption by mitochonria is suggested. Cytotoxicity of the germinated seeds extract was tested by the assessment of the viability, the morphological changes and the proliferation of cell cultured human skin fibroblasts. It has been shown that germinated seeds extract are non‐toxic to cell cultures at doses of up to 10 mg mL−1; neither morphological changes nor membrane integrity alterations or changes in cell proliferation were observed.

Structural organization of N-acyl derivatives of egg phosphatidylethanolamine into bilayers: polymorphic and thermotropic studies

Abstract The present paper focuses on the thermotropic and polymorphic behaviour of a series of N-acylphosphatidylethanolamines of natural origin. Headgroup and N-acyl chain modifications have been introduced, and the miscibility of the synthesized compounds with the main membrane phospholipids phosphatidylcholine and phosphatidylethanolamine has been studied. The compounds N-oleoylphosphatidylethanolamine and N-stearoylphosphatidylethanolamine of natural origin, and two different types of structural analogues have been synthesized and studied: N-oleoylphosphatidylpropanolamine with one extra methylene group between the phosphate and amino groups, and the N-alkyl derivative N-methyl-N-oleoylphosphatidylethanolamine. The N-acylation of transphosphatidylated phosphatidylethanolamine (PE) with saturated acyl chains caused a significant increase in the gel-to-liquid-crystalline phase transition temperature, determined by differential scanning calorimetric analysis, but all the N-oleoyl-containing glycerophospholipids assayed showed lower transition temperatures than their precursor aminophospholipids. The presence of a third hydrophobic chain, the possibility of intermolecular hydrogen bonding between the amide groups of N-acyl PEs and the loss of the hydrogen binding ability of aminophospholipids would account for this thermotropic behaviour. The miscibility of the N-acylphospholipids with egg phosphatidylcholine and transphosphatidylated egg yolk phosphatidylcholine has also been shown by these calorimetric studies, although non-ideal mixing of these phospholipid molecules has been revealed. 31P NMR studies have shown that the ability of the different N-acylated analogues to adopt lamellar structures is significantly different. When the N-acylation reactions were carried out with unsaturated acyl chains, the tendency to form nonlamellar phases was dramatically increased. Analysis of the 31P NMR spectra of mixtures of the different N-acylglycerophospholipids with the main membrane constituents phosphatidylcholine and phosphatidylethanolamine has shown that these lipid molecules are miscible, in agreement with the calorimetric data.

Nanostructured materials for photodynamic therapy: synthesis, characterization and in vitro activity

Three nanostructured vehicles are proposed as potential carriers for photosensitizers to be used in photodynamic therapy: spherical nanoparticles, hexahedral microparticles and cylindrical magnetic nanorods. A comparative study of their photodynamic properties was performed, and the influence of their size and the amount of loaded porphyrin was considered to discuss their effects in the observed photodynamic activity. All the vehicles have a gold surface, allowing functionalization with a disulfide-containing porphyrin as the photosensitizer, as well as with a PEG-containing thiol to improve their biocompatibility and water solubility. The activity of the porphyrin loaded in each vehicle was assessed through in vitro photocytotoxicity studies using HeLa cells. A synergic effect for the porphyrin toxicity was observed in all of the vehicles. The zinc-containing porphyrin showed better production of singlet oxygen, and proved more photocytotoxic both in solution and loaded in any of the vehicles. The magnetism of the nanorods allows targeting with a magnetic field, but causes their aggregation, hampering the porphyrins activity. Microparticles showed lower cell internalization but their bigger size allowed a high porphyrin loading, which translated into high photocytotoxicity. The highest cell internalization and photocytotoxicity was observed for the porphyrin-loaded nanoparticles, suggesting that a smaller size is favored in cell uptake.

Improved selectivity and cytotoxic effects of irinotecan via liposomal delivery: A comparative study on Hs68 and HeLa cells

&NA; Irinotecan (CPT‐11) is an effective chemotherapeutic agent widely used to treat different cancers. Otherwise, the liposomal delivery of anti‐tumor agents has been shown to be a promising strategy. The aim of this study has been to analyze the effect of liposomal CPT‐11 (CPT‐11lip) on two human cell lines (Hs68 and HeLa) to establish the suitability of this CPT‐11 nanocarrier. We have demonstrated the highest uptake of CPT‐11lip in comparison with that of CPT‐11sol, in lactate buffer, and that CPT‐11lip was internalized in the cells through an endocytic process whereas CPT‐11sol does so by passive diffusion. CPT‐11lip was not cytotoxic to normal fibroblast Hs68 cells, but induced a massive apoptosis accompanied by cell senescence in HeLa cells. CPT‐11lip treatment modified the morphology of HeLa cells, induced different cell cycle alterations and accumulated into lysosomes in both cell lines. In particular, CPT‐11lip treatment showed that surviving HeLa cells remained in a state of senescence whereas only a temporal growth arrest was induced in Hs68 cells. Results of RT‐PCR indicated that the different responses in Hs68 (survival) and HeLa cells (apoptotic death), seemed to be induced by a p53‐ and p53‐ independent mechanism, respectively. An analysis of DNA damage also determined that released CPT‐11 from liposomes was able to reach the nucleus and exert a genotoxic effect in both cell lines, which was repaired in Hs68 but not in HeLa cells. All results indicate that phospholipid‐cholesterol liposomes possess optimum properties for CPT‐11 delivery, being biocompatible and selectively cytotoxic against HeLa tumorigenic cells. Graphical abstract Figure. No caption available.

Gd-meso-tetraphenylporphyrins: synthesis, encapsulation into liposomes, and characterization of the resulting preparations

POPC, DPPC/DMPC and POPC/OOPS liposomes containing gadolinium(III) meso-tetraphenylporphyrin and gadolinium(III) meso-tetra(4-pyridyl)porphyrin have been prepared by different techniques, their porphyrin and lipid contents have been evaluated, and the size and stability of the liposomes have been determined. The results show the influence of the method of preparation on the amount of Gd-porphyrin incorporated, and that of the lipid composition on the incorporation efficiency and stability of the Gd(III) porphyrin trapped in the liposomes, as well as the presence of strong lipid to porphyrin association at least in the case of the DPPC/DMPC liposomes.

Permeability Studies of Liposomes of Phosphatidylcholine and N-(Oleoyl/Linoleoyl)-Phosphatidylhydroxyamines: Modification of the Head Group

The physical properties of N-acylethanolamine phospholipids have recently been reported. It has been shown that N-acylPEs are lipids which form closed lamellar structures in excess of water (Schmid et al., 1990). 31P-NMP spectroscopy, IR-FT, and freeze-fracture electron microscopy studies show the bilayer organization of the aqueous dispersions of these N-acyl derivatives and suggest the important physiological meaning of their synthesis from the non-bilayer phosphatidylethanolamine and from the membrane disturbing free fatty acids (Domingo et al., 1993; Domingo et al., 1994; Garcia et al., 1996). Biological membranes are barriers which selectively control the transfer of solutes in both directions and their permeability properties depend on its lipidic composition and on the physical state of them. Because of this, we have studied the consequences of the incorporation of different N-acyl-aminophospholipids into liposomes of egg phosphatidylcholine (EPC) and mixed phosphatidylcholine/cholesterol (Chol) with regard to the permeability characteristics of the resulting bilayers. The role of the head group structure of the N-acyl derivatives has been followed by modifying their hydrogen-bonding capacity and their steric bulk. Fluorescence spectroscopy has been the method used to establish one of the functional changes at the lipid bilayer level of biomembranes; that concerning their barrier properties.