Structuring and de-structuring of nanovectors from algal lipids. Part 1: physico-chemical characterization.

Lipid nanocarriers are among the most employed systems for drug delivery purposes in several research and industrial sectors, since their favorable properties ensure broad applicability. The design and characterization of these nanosystems are of paramount importance to obtain controlled outcome, since the supramolecular structure and molecular interactions deeply impact the functionality of the resulting aggregates. The choice of the most appropriate formulation for the target of interest relies on in-depth physico-chemical characterization in order to optimize stability, loading rates and sustained release. Several supramolecular architectures suited for carrier development can be obtained from lipid building blocks, by varying lipid composition and packing parameter. In particular, cubosome and liposome aggregates are often used as drug vectors thanks to their high cargo capability and biocompatibility. Moreover, the possibility to employ lipids from natural sources i.e. biomasses to prepare nanosystems makes them especially attractive. In this work, two aggregate types were characterized and compared as drug vectors for poorly water-soluble antioxidants, particularly curcumin and two adjuvants (i.e. tocopherol and piperine). The nanovectors were obtained by extracting lipids from algal biomasses with different lipid composition, and characterized by advanced structural (DLS, SAXS, Cryo-TEM) techniques, spectroscopy (NMR) and calorimetry (ITC). Finally, the structural stability of both aggregate types was evaluated.

Effect of quercetin, rutin, naringenin and epicatechin on lipid peroxidation induced in human sperm.

Quercetin, rutin, naringenin, epicatechin are flavonoids with diverse properties, including antioxidant potential. We evaluated, in vitro, the cytotoxicity of these flavonoids (20, 30, 50, 100, 200, 400 µM) in swim-up selected human sperm. Antioxidant activity was tested against tert-butylhydroperoxide induced lipid peroxidation using a C11-BODIPY(581/591) probe and transmission electron microscopy. A significant concentration-dependent effect on sperm viability (P<0.001) and motility (P<0.001) was observed. Lipid peroxidation was decreased in samples treated with 30 µM quercetin (P<0.01) and 30 µM rutin (P<0.05) versus samples incubated with tert-butylhydroperoxide alone. Naringenin (50-100 µM) showed a low protective effect and epicatechin (200 µM) was not efficacious. Transmission electron microscopy analysis confirmed the protective action of rutin and in particular quercetin on damages induced by lipid peroxidation. These results underlined the antioxidant properties of quercetin and rutin. A possible role of these compounds in the supplementation of media used during semen handling warrants attention and further studies.

Effect of trans-resveratrol on induced oxidative stress in human sperm and in rat germinal cells.

Resveratrol is a phytoalexin with antioxidant properties. We evaluated resveratrol toxicity in swim-up selected human sperm and in rat spermatocytes and spermatids separated by the STAPUT method. Resveratrol antioxidant activity was tested against lipid peroxidation (LPO) induced by tert-butyl hydroperoxide. LPO was evaluated using the C11-BODIPY(581/591) probe and transmission electron microscopy in samples incubated with and without resveratrol. LD50 for human sperm and rat spermatids was 50 µM; spermatocytes were more sensitive to resveratrol cytotoxicity. Sperm motility increased progressively at 30 µM, 15 µM and 6 µM. 15 µM resveratrol acts against LPO, preserving sperm chromatin and plasma membranes. LPO were more marked in spermatocytes than in spermatids and the effect of resveratrol was more evident in spermatocytes. In this study, the scavenger properties of resveratrol were demonstrated in vitro in human sperm and rat germ cells, thus resveratrol could be added to the media used in assisted reproduction techniques and cryopreservation when oxidative stress is exacerbated.

Antibacterial activity of grape extracts on cagA-positive and -negative Helicobacter pylori clinical isolates.

There is considerable interest in alternative/adjuvant approaches for the eradication of Helicobacter pylori using biologically active compounds, especially antioxidants from plants. In the present work, we tested the antioxidant and antimicrobial activities of hydro-alcoholic extracts from Colorino, Sangiovese and Cabernet Sauvignon grape cultivars against H. pylori G21 (cagA-negative, cagA-) and 10K, (cagApositive, cagA+) clinical isolates. We determined the minimum bactericidal concentration (MBC) by incubating strain suspensions in Brucella broth with fetal bovine serum and samples at different concentrations in a final volume of 100 microl in a microaerobic atmosphere. After incubation, subcultures were carried out on Brucella agar plates which were incubated for 3-5 days in a microaerobic environment. The lowest concentration in broth, where the subculture on agar showed complete absence of growth, was considered the MBC.The Colorino extract showed the highest antibacterial activity against G21 strain (MBC=1.35 mg/ml), while Sangiovese and Carbernet MBCs were 4.0 mg/ml ca. H. pylori 10K was only susceptible to Colorino after 48 hours (MBC = 3.57 mg/ml). Resveratrol exhibited the highest antibacterial activity. interestingly, the most pathogenic strain (10K) was less susceptible to both the grape extracts and the isolated compounds. These results suggest that the administration of grape extracts and wine constituents, in addition to antibiotics, might be useful in the treatment of H. pylori infection. Should the reduced susceptibility of 10K strain be extended to all the cagA+ H. pylori isolates, which are endowed with cancer promoter activity, this observation may help explain why the organisms expressing CagA are more closely associated with atrophic gastritis and gastric carcinoma development.

Nuclear magnetic resonance for studying recognition processes between anandamide and cannabinoid receptors.

The understanding of the molecular basis of cannabinoid activity has greatly improved since the discovery of CB1 and CB2 receptors. In this paper, the ligand binding processes between the endogenous cannabimimetic ligand, anandamide (AEA), and the cannabinoid receptors from different parts of rat brain were studied by nuclear magnetic resonance spectroscopy. The NMR approach is based on the comparison of selective (R1(SE)) and non-selective (R1(NS)) proton spin-lattice relaxation rates of the ligand in the presence and absence of macromolecular receptors, as well as R1(NS) and R1(SE) temperature dependency analysis. From these studies, the ligand-receptor binding strength was evaluated on the basis of the calculation of the "affinity index". The derivation of the "affinity index" from chemical equilibrium kinetics for all systems allowed the comparison of the ability of anandamide to interact with cannabinoid receptors present in different brain sectors.

Solution structure of folic acid. Molecular mechanics and NMR investigation.

The structure of folic acid in solution was investigated by nuclear magnetic resonance (NMR) and theoretical calculations. Dynamical information and geometrical constraints were obtained by carbon-13 relaxation study, homo-nuclear NOESY spectra and hetero-nuclear 1H-13C NOE experiments. This set of experimental data was used for the molecular mechanics and molecular dynamic calculations. The accuracy of the final structure was established by the R(NMR) factor, which was calculated comparing the experimental NOESY cross-peaks intensities and the corresponding values simulated by using the complete relaxation matrix analysis (CORMA) approach.

Solution structure of hyaluronic acid oligomers by experimental and theoretical NMR, and molecular dynamics simulation.

The conformational properties of hyaluronic acid (HA) oligomers in aqueous solution were investigated by combining high-resolution NMR experimental results, theoretical simulation of NMR two-dimensional (2D) spectra by Complete Relaxation Matrix Analysis (CORMA), and molecular dynamics calculations. New experimental findings recorded for the tetra- and hexasaccharides enabled the stiffness of the HA and its viscoelastic properties to be interpreted. In particular, rotating frame nuclear Overhauser effect spectroscopy spectra provided new information about the arrangement of the glycosidic linkage. From (13)C NMR relaxation the rotational correlation time (tau(c)) were determined. The tau(c) were employed in the calculation of geometrical constraints, by using the MARDIGRAS algorithm. Restrained simulated annealing and 1 ns of unrestrained molecular dynamic simulations were performed on the hexasaccharide in a box of 1215 water molecules. The beta(1 --> 3) and beta(1 --> 4) glycosidic links were found to be rigid. The lack of rotational degree of freedom is due to direct and/or water-mediated interresidue hydrogen bonding. Both single or tandem water bridges were found between carboxylate group and N-acetil group. The carboxylate group of glucuronic acid is not involved in a direct link with the amide group of N-acetyl glucosamine and this facilitated bonding between the residue and the water molecules.

Kinetic analysis and comparison of models of xylose metabolism by Klebsiella planticola.

A model for the degradation of xylose and ethanol production by Klebsiella planticola is proposed and compared with the exponential and Michaelis-Menten approaches. This model is based on the energy system diagrams and it is a simplified version of a previous model developed for the glucose and ethanol kinetics of the yeast Saccharomices cerevisiae. In this model the dynamics of the substrate and of the final product are strictly related by means of the cellular activity. This model shows superior performances with respect to the two alternatives, behaving better along the whole dynamics.