Mitochondrial DNA analysis of medieval sheep (Ovis aries) in central Italy reveals the predominance of haplogroup B already in the Middle Ages.

We retrieved 34 medieval ovicaprine remains, from three archaeological sites of central Italy dating to about 1000 years old, and analyzed them using mitochondrial DNA. We compared the reconstructed haplogroups with modern sheep samples from Europe and the Middle East and sequences from the literature. In modern sheep, haplogroup HA is present in countries with access to the Mediterranean and close to the domestication center, whereas it is very rare or absent in the rest of Europe. The haplogroup HB was predominant in ancient samples (90%), whereas haplogroup HA was found at 10%. Ancient haplogroups match the present distribution in modern sheep in Italy, indicating that the current proportion of HA/HB was already established in the Middle Ages and is not the result of subsequent events such as selective breeding practices.

Gel-embedded niosomes: preparation, characterization and release studies of a new system for topical drug delivery.

In the present paper physical gels, prepared with two polysaccharides, Xanthan and Locust Bean Gum, and loaded with non-ionic surfactant vesicles, are described. The vesicles, composed by Tween20 and cholesterol or by Tween85 and Span20, were loaded with Monoammonium glycyrrhizinate for release experiments. Size and zeta (ζ)-potential of the vesicles were evaluated and the new systems were characterized by rheological and dynamo-mechanical measurements. For an appropriate comparison, a Carbopol gel and a commercial gel for topical applications were also tested. The new formulations showed mechanical properties comparable with those of the commercial product indicating their suitability for topical applications. In vitro release experiments showed that the polysaccharide network protects the integrity of the vesicles and leads to their slow release without disruption of the aggregated structures. Furthermore, being the vesicles composed of molecules possessing enhancing properties, the permeation of the loaded drugs topically delivered can be improved. Thus, the new systems combine the advantages of matrices for a modified release (polymeric component) and those of an easier permeability across the skin (vesicle components). Finally, shelf live experiments indicated that the tested gel/vesicle formulations were stable over 1 year with no need of preservatives.

Synthesis and characterization of N-(2-aminoethyl)-2-acetamidyl gellan gum with potential biomedical applications.

N-(2-aminoethyl)-2-acetamidyl gellan gum (GCM-EDA) was prepared by carboxymethylation (via nucleophilic substitution of primary hydroxyl groups of the ß-D-glucose unit of gellan gum, in the presence of alkali and chloroacetic acid) and reaction with tert-butyl N-(2-aminoethyl) carbamate (N-Boc-EDA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as an activator, followed by deprotection with trifluoroacetic acid. The structural confirmation and characterization of N-(2-aminoethyl)-2-acetamidyl gellan gum was performed by spectroscopic, rheological and thermogravimetric analysis, and in vitro tests showed a lack of cytotoxicity which is indicative of the potential of this material to be used in biomedical applications.

Injectable and in situ gelling hydrogels for modified protein release.

A novel injectable polysaccharide system based on calcium Alginate (Ca-Alg) hydrogel and two Dextran methacrylate derivatives (DexMA) was recently developed. The resulting Interpenetrating Polymer Network showed a synergistic mechanical behavior that can be exploited to target the hydrogel properties towards specific biomedical needs. In the present paper, hydrogels composed of 3% (w/v) Ca-Alg and Dextran (Mw 40 x 10(4) and 500 x 10(4)), derivatized with methacrylic groups (derivatization degrees 5 and 30%) at concentrations 5% (w/v), were characterized. The data reported here evidenced that Mw and derivatization degree of Dex chains can deeply affect the mechanical as well a model protein (Horseradish peroxidase) delivery rate. The enzymatic activity of such model protein was never significantly altered by the adopted experimental conditions.

pH-sensitive non-phospholipid vesicle and macrophage-like cells: binding, uptake and endocytotic pathway.

Phospholipid and non-phospholipid vesicles are extensively studied as drug delivery systems to modify pharmacokinetics of drugs and to improve their action in target cells. It is believed that the major barrier to efficient drug delivery is entrapment of drugs in the endosomal compartment, since this eventually leads to its degradation in lysosomes. For these reasons, the knowledge of internalization pathway plays a fundamental role in optimizing drug targeting. The aim of this work is to characterize pH-sensitive Tween 20 vesicles, their interaction with macrophage-like cells and their comparison with pH-sensitive liposomes. The effect of different amounts of cholesteryl hemissucinate on surfactant vesicle formation and pH-sensitivity was studied. To evaluate the initial mode of internalization in Raw 264.7 and the intracellular fate of neutral and pH-sensitive formulations, flow cytometry in presence and in absence of selected inhibitors and fluorescence microscopy in absence and presence of specific fluorescent endocytotic markers were used. The obtained results showed that the surfactant vesicle pH-sensitivity was about two or three fold higher than that obtained with pH-sensitive liposomes in the presence of serum in vitro. The uptake mechanism of surfactant vesicles, after incubation with macrophage-like cells, is comparable to that of liposomes (clathrin-mediated endocytosis).

Characterization of polysaccharide hydrogels for modified drug delivery.

Hydrogels are hydrophilic macromolecular networks that are capable of retaining a large amount of water. A precise description of these systems is actually quite complex and the practical use of hydrogels for drug delivery and biomedical applications is often not supported by a well-defined knowledge of the overall structure of the polymeric network. In this paper, we report the characterization of two different systems: a chemical network based on Guar Gum (GG) and a physical gel prepared with Xanthan (Xanth) and Locust Bean Gum (LBG). The dynamo-mechanical properties of the gels were analysed: the cohesiveness and the adhesion of the networks were strongly dependent on time, temperature, and composition. The kinetics of the chemical crosslinking was followed by means of rheological measurements, i.e. recording the mechanical spectra of the gelling system, and the power law exponent at the gel point was evaluated. Furthermore, the networks, loaded with model drugs with different steric hindrance, were used as matrices for tablets and the rate of release of such model drugs was studied. The diffusion of the guest molecules was deeply dependent on their dimensions; in the case of Xanth-LBG tablets the release profiles were almost independent from the different cohesion properties of the starting hydrogel composition.

Scleroglucan/borax: characterization of a novel hydrogel system suitable for drug delivery.

A new hydrogel, with scleroglucan using borax as a crosslinker, has been prepared. The physical gel has been loaded with a model molecule (theophylline) and the release of the drug from the gel was evaluated. The same system was used to prepare tablets and the delivery of theophylline in different environmental conditions (HCl and SIF) was determined. A recent theoretical approach has been applied to the dissolution profiles obtained from the tablets and a satisfactory agreement has been found with the experimental data. Furthermore, the diffusion coefficient of the model molecule was evaluated according to a suitable strategy that was tested on two set of data obtained with different set-ups (permeation and diffusion experiments). A simplified mathematical approach allows to reduce the two-dimensional problem of the Fick's second law in a one-dimensional system leading to a much easier handling of the data without loosing the accuracy of the original problem in two dimensions. The characterization of the gel has been also carried out following the kinetics of swelling in terms of water uptake.

A crosslinked system from scleroglucan derivative: preparation and characterization.

Matrices obtained by a crosslinking reaction between the polycarboxylated derivative of scleroglucan (sclerox) and 1,6-hexanedibromide have been prepared and characterized. Different ratios between the alkane dihalide and sclerox yielded products with different properties. Water uptake by the hydrogel with a low degree of crosslinking was remarkably affected by ionic strength. The determination of the crosslink density is led by simultaneously solving two Flory equilibrium equations referring to two different conditions characterized by the presence or the absence of a salt in the swelling agent. Moreover, the swelling kinetics was studied by means of a recently proposed model. Finally, the permeation of two model molecules (theophylline and polystyrene sulphonate-sodium salt) through the hydrogels was evaluated.

Low-degree oxidized scleroglucan and its hydrogel.

A controlled oxidation of scleroglucan was performed with sodium periodate to prepare aldehyde derivatives (scleraldehyde) with a low degree of oxidation (10 and 20%), which were utilized for crosslinking reactions with hexamethylenediamine. The structural characterization of scleraldehydes and their corresponding hydrogels was attempted by small-angle X-ray scattering (SAXS). While scleraldehyde with a higher degree of oxidation (> or = 50%), according to an earlier research, was found to disentangle into single chains as the degree of oxidation increases; scleroglucan bearing a low percentage of aldehydic groups (up to 20%) retains mainly the conformation of the natural polysaccharide, thus the system can be represented as composed of triple helices with only minor disentanglements at the sites where the aldehyde groups are present. The hydrogel prepared from scleraldehyde with a low degree of oxidation is brittle and fragmented, in contrast to the elastic/homogeneous hydrogel earlier prepared from scleraldehyde with a high degree of oxidation. The hydrogel from scleraldehyde with a low degree of oxidation was found to possess a network structure that consisted mostly of the triple helices crosslinked in specific points where the triple helices are disentangled into single chains because of the presence of the aldehyde groups.

Development of a profitable procedure for the extraction of 2-H-1-benzopyran-2-one (coumarin) from Mikania glomerata.

This report deals with a new procedure suitable for the extraction of coumarin 1 from Mikania glomerata. The aim of this investigation is to obtain this compound in an economically profitable way, taking into account the yield of its extraction, the cost, and the time of the overall process. Fresh and dried plants collected in several areas of the State of Rio de Janeiro were used, and seasonal effects on coumarin content were studied. Obtained results indicated that extraction with a 1% (w/v) NaOH solution, under appropriate conditions, allows a simple and complete recovery of the desired product and that the best yields were obtained with the fresh aerial parts of the plant. Season and area of harvesting effects have also been studied.

Novel hydrogel system from scleroglucan: synthesis and characterization.

New hydrogels obtained by a crosslinking reaction between the polycarboxylated derivative of scleroglucan (sclerox) and 1, 6-hexanedibromide have been prepared and characterized. Different ratios between the alkane dihalide and sclerox yielded products with appreciably different properties. Water uptake by the hydrogel with a low degree of crosslinking was remarkably affected by ionic strength. The diffusion of a model molecule (theophylline) through the swelled crosslinked polymers was studied and the theoretical analysis leading to the calculation of permeability coefficients in different environmental conditions is reported. Tablets prepared with one of the new hydrogels behaved as swellable monolithic systems suitable for sustained drug release.

Effects of surfactants on the spectral behaviour of calcein (II): a method of evaluation.

The spectral behavior of calcein, a water-soluble self quenching fluorescent marker often used in biomedical analysis, can be considerably affected by the presence of surfactants. With this study we intend to obtain further information on the photophysical properties of calcein, in the presence of surfactants and in the concentration range commonly used to investigate the release of such marker from vesicle dispersions. The experiments were carried out both in water and in a physiological buffer (HEPES, pH 7.5), in the presence of Triton X-100, sodium dodecyl sulphate and centyltrimethylammonium bromide, both below and above their critical micelle concentration (c.m.c.). The obtained results confirm that calcein fluorescence can be affected by the presence of surfactants. Thus, environmental conditions must always be carefully checked for the actual quantitative evaluation of this dye. Furthermore, this study sheds some light on the nature and mechanism of calcein quenching.

A novel co-crosslinked polysaccharide: studies for a controlled delivery matrix.

The formulation of a new controlled delivery system, based on a novel type of matrix obtained by the chemical reaction carried out in an aqueous medium on a mixed physical gel of gellan and scleroglucan, is described in this paper. The preparation yielded a new co-crosslinked polysaccharide (CCP) hydrogel, bearing carboxylic groups, that showed a sustained release behaviour that can be modulated by means of calcium ions. For the characterization of CCP, diffusion experiments through the swelled hydrogel were carried out in different environmental conditions and the release from tablets prepared with CCP and a model drug was evaluated. The addition of CaCl2 in the formulation of the dosage forms allowed a further marked reduction in delivery rate to be obtained; this effect is to be related to the free ionized carboxylic groups still present in the gellan moiety of CCP. The different behaviour of Ca+2 and Na+ ions is discussed.

Effects of in-vitro activity of miconazole and ketoconazole in phospholipid formulations.

Antifungal agents are often used in liposomal formulations in order to improve their pharmacological activity, but how vesicle inclusion can actually affect this is still not fully understood. We report here the results obtained from evaluation of the in-vitro activity against Candida albicans ATCC E10231 of miconazole and ketoconazole in various vesicular and non-vesicular preparations, obtained from egg and soya phospholipids, using time-kill curves. In most cases inclusion of miconazole or ketoconazole in liposomes led to a delayed and decreased activity of the drugs, with detectable differences among the various phospholipid concentrations and different liposomal preparations (small unilamellar vesicle, liposomes, multilamellar aggregates and broken liposomal structures). The results obtained may be helpful in the study of new preparations of antifungal agents entrapped in liposomal structures.

Gellan in sustained release formulations: preparation of gel capsules and release studies.

The ability of gellan to form gels in the presence of calcium ions enabled us to prepare capsules by gelation of this polysaccharide around a core containing starch, calcium chloride and a model drug. Release from the dried capsules was studied in vitro by means of the rotating basket technique (USP) in different environmental conditions (distilled water, pH = 2.0, pH = 6.8) and the effects of the presence of increasing amounts of drug in the formulation were also investigated. The behaviour of the gellan capsules was compared with that of beads prepared with the same polysaccharide but containing different additives. Results obtained indicate that gellan is suitable for the formulation of sustained release capsules and that solvent uptake by the dried capsules is most likely the main factor capable of affecting the rate of delivery from the tested preparations.

Effects of surfactants on the spectral behaviour of calcein.

The spectral behaviour of calcein, a water-soluble self-quenching molecule that is often used as a marker in biomedical analysis, can be considerably affected by the presence of surfactants. The aim of this work was to investigate the spectral properties of calcein under conditions that are particularly significant in liposome studies. For this purpose the fluorescence and absorbance of this dye were determined in solutions of ionic and non-ionic surfactants (cetyltrimethylammonium bromide, sodium dodecyl sulphate and Triton X-100), at different concentrations below and above their critical micelle concentrations (c.m.c.) as well as in the presence of phospholipids in the form of liposomes and/or mixed micelles. Cationic surfactants induced changes in lambda max, absorbance and fluorescence but these changes were less noticeable in the phospholipid dispersions. The anionic and the non-ionic surfactants induced mainly changes in fluorescence intensity.

Effect of surfactants on diffusion of calcium in mucin: a possible mechanism affecting enamel remineralization.

By means of in vitro experiments, performed with a three compartment diffusion cell, we show how the presence of surfactants, often present in the formulation of dentifrices and mouthrinses, may affect the availability of calcium and its permeation rate through the proteic layer covering dental enamel (acquired pellicle). Mucin was the protein used to simulate the pellicle. Experiments were performed at different surfactant concentrations (i.s. below and above CMC) and also in the presence of fluoride ions that are capable of enhancing enamel remineralization. The mechanisms involved, mainly related to the interaction of surfactant monomers with the model protein, are discussed in this paper.

Scleroglucan sustained release oral preparations. Part II. Effects of additives.

Swelling and diffusion experiments, performed in vitro with tablets prepared with scleroglucan and several hydrophilic and hydrophobic additives, indicate that it is possible to modulate drug delivery from the matrix by appropriate choice of the nature and amount of the additive. The additives in the formulations may affect the mechanisms (zero order, diffusion, erosion) involved in the release of drugs from the dosage forms.