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Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length

Daniele Rubert Nogueira, Montserrat Mitjans, M. Carmen Morán, Lourdes Pérez, M. Pilar Vinardell

Amino Acids, 43, 1203–1215, 2012

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of Ne- and Na-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the a-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.


Protein-covered silica nano-particles adsorbing onto synthetic vesicles

Federica De Persiis, Camillo La Mesa and Ramon Pons

Soft Matter, 8, 1361, 2012

Silica nano-particles with surface acid groups were reacted with coupling agents and, then, with lysozyme, to get functionalized entities. The procedure functionalizes surface sites and ensures moderate coverage. The amount of bound lysozyme, estimated from UV-vis, is 5 molecules per nanoparticle. Electro-phoretic mobility indicates variations in surface charge density of functionalized nanoparticles compared to the original ones. Protein-functionalized nano-particles form clusters, redispersible by application of shear. Such nano-particles interact with vesicles comparable in size. The kinetic pathways of the interactions between them were investigated. Adsorption onto vesicles and clustering are diffusion controlled. The process is governed by repulsive and attractive interactions between such entities. Nano-particle/vesicle adducts precipitate as fine powders, or form large floating objects, depending on vesicle size, charge, and [vesicles/nano-particles] ratios. Attempts were made to rationalize such behavior as the combination of electro-phoretic mobility and diffusive contributions of the interacting species.


Bicelles: Lipid Nanostructured Platforms with Potential Dermal Applications

Lucyanna Barbosa-Barros, Gelen Rodríguez, Clara Barba, Mercedes Cócera, Laia Rubio, Joan Estelrich, Carmen López-Iglesias, Alfonso de la Maza, and Olga López

small, 8, (6), 807–818, 2012

Bicelles emerge as promising membrane models, and because of their attractive combination of lipid composition, small size and morphological versatility, they become new targets in skin research. Bicelles are able to modify skin biophysical parameters and modulate the skin's barrier function, acting to enhance drug penetration. Because of their nanostructured assemblies, bicelles have the ability to penetrate through the narrow intercellular spaces of the stratum corneum of the skin to reinforce its lipid lamellae. The bicelle structure also allows for the incorporation of different molecules that can be carried through the skin layers. All of these characteristics can be modulated by varying the lipid composition and experimental conditions. The remarkable versatility of bicelles is their most important characteristic, which makes their use possible in various fields. This system represents a platform for dermal applications. In this review, an overview of the main properties of bicelles and their effects on the skin are presented.


New identification of proanthocyanidins in cinnamon (Cinnamomum zeylanicum L.) using MALDI-TOF/TOF mass spectrometry

María Luisa Mateos-Martín, Elisabet Fuguet, Carmen Quero, Jara Pérez-Jiménez, Josep Lluís Torres

Anal Bioanal Chem 402, 1327–1336, 2012

The inner bark of Ceylon cinnamon (Cinnamomum zeylanicum L.) is commonly used as a spice and has also been widely employed in the treatment and prevention of disease. The positive health effects associated with the consumption of cinnamon could in part be due to its phenolic composition; proanthocyanidins (PA) are the major polyphenolic component in commercial cinnamon. We present a thorough study of the PA profile of cinnamon obtained using matrix-assisted laser desorption/ionization tandemtime-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition to the advantages of MALDI-TOF as a sensitive technique for the analysis of high-molecular-weight compounds, the tandem arrangement allows the identification of the compounds through their fragmentation patterns from MS/ MS experiments. This is the first time that this technique has been used to analyze polymeric PA. The results show that cinnamon PA are more complex than was previously thought. We show here for the first time that they contain (epi)gallocatechin and (epi)catechingallate units. As gallates (galloyl moieties) and the pyrogallol group in gallocatechins have been related to the biological activity of grape and tea polyphenols, the presence of these substructures may explain some of the properties of cinnamon extracts. MALDI-TOF/ TOF reveals that cinnamon bark PA include combinations of (epi)catechin, (epi)catechingallate, (epi)gallocatechin, and (epi)afzelechin, which results in a highly heterogeneous mixture of procyanidins, prodelphinidins, and propelargonidins.


Proposed Bioactive Conformations of Opiorphin, an Endogenous Dual APN/NEP Inhibitor

Marta Pinto, Catherine Rougeot, Luis Gracia, Monica Rosa, Andres Garcıa , Gemma Arsequell, Gregorio Valencia, and Nuria B. Centeno

ACS Med. Chem. Lett. 3, 20−24, 2012

The conformational profiles for the endogenous peptide Opiorphin and a set of seven analogues exhibiting different inhibitory activities toward human aminopeptidase N (hAPN) and human neprilysin (hNEP) were independently computed to deduce a bioactive conformation that Opiorphin may adopt when binding these two enzymes. The conformational space was thoroughly sampled using an iterative simulated annealing protocol, and a library of low-energy conformers was generated for each peptide. Bioactive Opiorphin conformations fitting our experimental structure−activity relationship data were identified for hAPN and hNEP using computational pairwise comparisons between each of the unique low-energy conformations of Opiorphin and its analogues. The obtained results provide a structural explanation for the dual hAPN and hNEP inhibitory activity of Opiorphin and show that the inborn flexibility of Opiorphin is essential for its analgesic activity.


Photodamage determination of human hair

Estibalitz Fernández, Clara Barba, Cristina Alonso, Meritxell Martí, José Luis Parra, Luisa Coderch

Journal of Photochemistry and Photobiology B: Biology 106, 101–106, 2012

Sunlight on human hair causes photo-degradation. This results in bleaching due to melanin oxidation through free radicals, and induces keratin impairment. Protein degradation, tryptophan degradation, lipidic peroxidation and electron paramagnetic resonance can be used to evaluate proteic and lipidic photodecomposition and free radical formation in hair fibres subjected to antioxidant action and different UV intensities. All these methodologies have been optimised to determine protein, lipid and melanin degradation in hair subjected to different UV intensities.


Non-extractable proanthocyanidins from grapes are a source of bioavailable (epi)catechin and derived metabolites in rats

María Luisa Mateos-Martín, Jara Pérez-Jiménez, Elisabet Fuguet, and Josep Lluís Torres

Brt. J.Nutr., 108, 290-297, 2012

The non-extractable fraction of many fruit and vegetables contains putatively bioactive polyphenolic compounds that, in most cases, have not been well characterised structurally. Non-extractable proanthocyanidins (NEPA) of a polymeric nature are part of the dietary fibre fraction of food. Using liquid chromatography coupled to a mass spectrometer equipped with an electrospray ionisation chamber and a triple quadrupole mass analyser for tandem analysis (HPLC–ESI–QqQ–MS/MS) techniques, we examine the phenolic metabolites present in urine and faeces from rats 24 h after ingestion of an NEPA-rich fraction. We show that NEPA are partially depolymerised during their transit along the intestinal tract, as evidenced by the presence of (epi)catechin (EC) monomers and dimers in faeces and phase II conjugates of EC in urine. Moreover, NEPA are further metabolised by the intestinal microbiota into smaller metabolites including phenolic acids that are present in urine as both free phenolics and conjugates with glucuronate or sulphate moieties. For the first time, we report evidence that NEPA behave in vivo as a source of phenolics that are released progressively and deliver phenolic species that come into contact with the intestinal walls and are bioavailable for at least 24 h after ingestion.


Generation of a Human Neuronal Stable Cell Model for Niemann-Pick C Disease by RNA Interference

Laura Rodríguez-Pascau, Maria Josep Coll, Josefina Casas, Lluïsa Vilageliu, and Daniel Grinberg

JIMD Rep., 4, 29–37, 2012

Niemann–Pick type C (NPC) disease is a fatal autosomal recessive neurodegenerative disorder caused, most commonly, by mutations in the NPC1 gene. At the cellular level, the disease is characterized by the storage of multiple lipids in the endosomal–lysosomal system, including free cholesterol, glycosphingolipids, sphingomyelin and the catabolic product of sphingolipids, sphingosine. Therapeutic options for NPC disease are relatively limited. One drawback for the development of novel therapies is the lack of suitable human neuronal cell models. In this work, a stable SH-SY5Y cell model for NPC disease was generated using short hairpin RNAs. An inhibition of the NPC1 expression of around 90% was obtained at the RNA level. The NPC1 knockdown was confirmed at the protein level. To characterize the stable cell line generated, cholesterol levels were analyzed in the NPC1-knockdown SH-SY5Y cells by filipin staining and gas chromatography–mass spectrometry. A characteristic NPC pattern and a twofold increase of the free cholesterol levels, related to intact SH-SY5Y cells, were found. Moreover, sphingolipids were analyzed by liquid chromatography–mass spectrometry and an increase in ganglioside GM2 levels was observed. The stable NPC1-knockdown SH-SY5Y cell line generated in the present study provides a human neuronal cell model for this lethal disease that could be a valuable tool for the study of future therapeutic approaches.


D-Fagomine lowers postprandial blood glucose and modulates bacterial adhesion

Livia Gómez, Eunice Molinar-Toribio, María Ángeles Calvo-Torras, Carles Adelantado, M. Emília Juan, Joana M. Planas, Xavier Cañas, Carles Lozano, Sergio Pumarola, Pere Clapés and Josep Lluís Torres

British Journal of Nutrition, 107, 1739–1746, 2012

D-Fagomine is an iminosugar originally isolated from seeds of buckwheat (Fagopyrum sculentum Moench), present in the human diet and now available as a pure crystalline product. We tested D-fagomine for activities connected to a reduction in the risk of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. The activities were: intestinal sucrase inhibition in vitro (rat mucosa and everted intestine sleeves), modulation of postprandial blood glucose in rats, bacterial agglutination and bacterial adhesion to pig intestinal mucosa. When ingested together with sucrose or starch, D-fagomine lowered blood glucose in a dose-dependent manner without stimulating insulin secretion. D-Fagomine reduced the area under the curve (0–120 min) by 20% (P<0·01) and shifted the time to maximum blood glucose concentration (Tmax) by 15 min at doses of 1–2 mg/kg body weight when administered together with 1 g sucrose/kg body weight. Moreover, D-fagomine (0·14mM) agglutinated 60% of Enterobacteriaceae (Escherichia coli, Salmonella enterica serovar Typhimurium) populations (P<0·01), while it did not show this effect on Bifidobacterium spp. or Lactobacillus spp. At the same concentration, D-fagomine significantly (P<0·001) inhibited the adhesion of Enterobacteriaceae (95–99% cells in the supernatant) and promoted the adhesion of Lactobacillus acidophilus (56% cells in the supernatant) to intestinal mucosa. D-Fagomine did not show any effect on bacterial cell viability. Based on all this evidence, D-fagomine may be used as a dietary ingredient or functional food component to reduce the health risks associated with an excessive intake of fast-digestible carbohydrates, or an excess of potentially pathogenic bacteria.


C6-Ceramide and targeted inhibition of acid ceramidase induce synergistic decreases in breast cancer cell growth

Margaret Flowers, Gemma Fabriás, Antonio Delgado, Josefina Casas, Jose Luis Abad, Myles C. Cabot

Breast Cancer Res Treat, 133, 447–458, 2012

The sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death. Acid ceramidase (AC) hydrolyzes ceramide, and thus reduces intracellular levels of this proapoptotic lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in prostate cancer and in some breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosisinducing effects of C6-ceramide (C6-cer) in breast cancer cells. Cultured breast cancer cells were treated with DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of reactive oxygen species, and mitochondrial transmembrane potential were determined. DM102 was a more effective AC inhibitor than N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(40-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC50 5–10 lM) and DM102 (IC50 20 lM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index\0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of caspase 3 cleavage and a[3-fold increase in caspase 3/7 activation, PARP cleavage, and a[70% increase in Annexin-V positive cells. C6-cer/DM102 increased ROS levels 4-fold in MDAMB-231 cells, shifted the ratio of Bax:Bcl-2 to[9-fold that of control cells, and resulted in mitochondrial membrane depolarization. DM102 also increased the synthesis of 3Hpalmitate-labeled long-chain ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/DM102 drug duo in human breast cancer.


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