Human hepatoma cell lines on gas foaming templated alginate scaffolds for in vitro drug-drug interaction and metabolism studies.

Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was also observed when the more differentiated HepaRG cells were used. Together the data suggest that hepatocyte/gas templated alginate-systems provide an innovative high throughput platform for in vitro drug metabolism and drug-drug interaction studies, with broad fields of application, and might provide a valid tool for minimizing animal use in preclinical testing of human relevance.

Anomalous Debye-like dielectric relaxation of water in micro-sized confined polymeric systems.

While it is well known that spatial confinement on a nm scale affects the molecular dynamics of water resulting in a hindered dipolar reorientation, question of whether these effects could result at length scales larger than these, i.e., in confined regions of the order of µm or more, is still under debate. Here we use dielectric relaxation spectroscopy techniques to study the relaxation orientation dynamics of water entrapped in different polymeric matrices with pore sizes of the order of 100 µm, analyzing the frequency relaxation behaviour of the dielectric response. Our results show that, contrary to what has been generally thought, even in confinements which are not particularly high such as those realized here, regions typically hundred micrometers in size can affect the water structure, inducing a water phase with properties different from those of bulk water. In particular, we observe a dielectric dispersion centered in the range 10(5)-10(7) Hz, in between the one characteristic of ice (8.3 kHz at T = 0 °C) and the one of bulk water (19.2 GHz at T = 25 °C). The analysis of the dependence on temperature of the relaxation time of this unexpected contribution rules out the possibility that it can be attributed to an interfacial polarization (Maxwell-Wagner effect) and suggests a dipolar Debye-like origin due to a slow-down of the hydrogen-bonded network orientational polarization. Also at these scales, the confinement alters the structure of water, leading to a hindered reorientation. These properties imply that water confined within these polymeric porous matrices is more ordered than bulk water. These findings may be important in order to understand biological processes in cells and in different biological compartments, where water is physiologically confined.

Dielectric characterization of hepatocytes in suspension and embedded into two different polymeric scaffolds.

The dielectric and conductometric properties of hepatocytes in two different environments (in aqueous suspension and embedded into polymeric scaffolds) have been investigated in the frequency range from 1 kHz to 2 GHz, where the interfacial electrical polarization gives rise to marked dielectric relaxation effects. We analyzed the dielectric behavior of hepatocytes in complete medium aqueous suspensions in the light of effective medium approximation for heterogeneous systems and hepatocytes cultured into two different highly porous and interconnected polymeric structures. In the former case, we have evaluated the passive electrical parameters associated with both the plasmatic and nuclear membrane, finding a general agreement with the values reported elsewhere, based on a partially different analysis of the experimental spectra. In the latter case, we have evaluated the cell growth into two different polymeric scaffolds made of alginate and gelatin with a similar pore distribution and similar inter-connectivity. Based on a qualitative analysis of the dielectric spectra, we were able to provide evidence that alginate scaffolds allow an overall survival of cells better than gelatin scaffold can do. These indications, confirmed by biological tests on cell viability, suggest that hepatocytes embedded in alginate scaffolds are able to perform liver specific functions even over on extended period of time.

A biomimetic porous hydrogel of gelatin and glycosaminoglycans cross-linked with transglutaminase and its application in the culture of hepatocytes.

The development of blended gelatin and glycosaminoglycan (GAG) scaffolds can potentially be used in many soft tissue engineering applications since these scaffolds mimic the structure and biological function of native extracellular matrix (ECM). In this study, we were able to obtain a gelatin-GAG scaffold by using a concentrated emulsion templating technique known as high internal phase emulsion (HIPE), in which a prevailing in volume organic phase is dispersed in the form of discrete droplets inside an aqueous solution of three biopolymers represented by gelatin, hyaluronic acid (HA) and chondroitin sulfate (CS) in the presence of a suitable surfactant. In order to preserve the bioactive potential of the biopolymers employed, the cross-linking procedure involved the use of transglutaminase (MTGase) that catalyzes the formation of covalent N-ε-(γ-glutamyl) lysine amide bonds. Since neither HA nor CS possess the necessary primary amino groups toward which MTGase is active, they were functionalized with the dipeptide glycine-lysine (GK). In this way the introduction of foreign cross-linking bridging units with an unpredictable biocompatibility was avoided. These enzymatic cross-linked gelatin-GAG scaffolds were tested in the culture of primary rat and C3A hepatocytes. Results underlined the good performance of this novel support in maintaining and promoting hepatocyte functions in vitro.

PLGA-based nanoparticles: effect of chitosan in the aggregate stabilization. A dielectric relaxation spectroscopy study.

Chitosan-modified polylactic-co-glycolic acid (PLGA) nanoparticles with average diameter of 200 nm in PBS buffer solution have been investigated by means of dielectric relaxation spectroscopy measurements in the frequency range (1 MHz-2 GHz) where interfacial polarizations occur. PLGA-based nanoparticles offer remarkable advantages in different biotechnological fields, such as their biocompatibility, easiness of administration and rather complete biodegradation. However, despite the use of these drug delivery systems is increasing, little is known about the basic process involved in the formation of complexes and in the subsequent release kinetics. In the present work, we have characterized the colloidal behavior of PLGA-based nanoparticles in the presence of oppositely charged chitosan polyelectrolyte by means of dynamic light scattering, electrophoretic mobility and radiowave dielectric relaxation measurements. In particular, we have emphasized how the presence of a coating layer at the nanoparticle surface could exert a marked slowing-down in the drug release. The consequence of this finding is briefly discussed at the light of some biological implications.

Alginate beads as immobilization matrix for hepatocytes perfused in a bioreactor: a physico-chemical characterization.

Because of their peculiar physico-chemical properties, alginate beads have often been proposed as an alternative cell immobilization matrix for many biotechnological applications. For entrapped hepatocytes perfused in a bioreactor, alginate beads have been demonstrated to promote viability and three-dimensional cell organization. In order to optimise the hepatocyte cell culture, we investigated the relationship between alginate beads properties, at high and low content of guluronic acid (G), and the relative cell viability and reorganization when perfused in a bioreactor. The primary structure of alginates did not apparently influence the hepatocytes culture in 8 h of perfusion in a bioreactor. However, our results confirm a preference for beads with a high content of G due to their superior mechanical resistance.

Enzymatic epimerization of bacterial mannuronan and of C-6 oxidized, galactose-depleted guar: a circular dichroism and 1H NMR study.

Attention has been focused on two uronans, namely, mannuronan and galactose-depleted C-6 oxidized guar, the former of microbial origin and the latter of artificial nature, to provide original data on the extent of epimerization they can undergo in dilute aqueous solution using two C-5 mannuronic acid epimerizing enzymes, that is, AlgE-4 and AlgE-6, alone or in admixture. Original circular dichroism data coupled with 1H NMR spectra clearly point out that both uronans can be epimerized, depending on the enzyme or enzyme mixture used, to high levels yielding guluronic-rich alginate samples and guluronic-rich heteropolysaccharides, respectively. Mannuronan and its epimerization products can easily form clear, firm aqueous gels when an excess of HCl is added or when mixed with aqueous CaCl2, respectively. Depleted-guarox does not gel upon addition of excess HCl, while the heterouronan derived from it having a percent of epimerization nearly identical to that of epimerized mannuronan, that is, ca. 70%, can form gel in the presence of Ca(II) only at higher polymer and Ca(II) concentrations. With the latter, heterouronan alpha-D-galacturonic side groups exert hindrance to "junction zone" formation.

Macromolecular triplex zipping observed in derivatives of fungal (1 --> 3)-beta-D-glucan by electron and atomic force microscopy.

Scleroglucan, a comb-like branched (1 --> 3)-beta-D-glucan, dissolves in water as a stiff, triple-helical structure with the single glucose branches extending from the surface. The aim of this study is to investigate structural changes in the triple-helical structure associated with selective chemical modification of the side chains. Electron and atomic force microscopy, respectively, were used to investigate the macromolecular structures of aldehyde and carboxylated derivatives of scleroglucan-namely, scleraldehyde and sclerox-with different degrees of substitution. Scleraldehyde was observed to have structures resembling the triplex of the unmodified scleroglucan for all degrees of substitution up to 1.0. Additionally, an increasing tendency to aggregate for the higher degrees of substitution was observed. Fully carboxylated scleroglucan, sclerox(1.0), prepared from solutions at ionic strengths below 1.0M, revealed dispersed, flexible, coil-like structures. This indicates an electrostatic-driven strand separation of the scleroglucan triple-helical structure occurring concomitant with an increasing fraction of the side chains bearing carboxylate groups. Annealed sclerox(1.0) samples in aqueous 1.0 and 1.5M NaCl exhibited partly, or completely, reassociated triplex ensembles, with species ranging from apparently fully zipped linear and circular topologies, partly zipped structures with triplex strand separation occurring at the ends, to dispersed single-strands with random coil-like appearance. This study shows that periodate oxidation of the scleroglucan side chains is not a sufficient modification of the side chains to induce dissociation of the triple-helical structure, whereas further oxidation of the side chains to carboxylic groups dissociates the triple-helical structure when the degree of substitution is above 0.6.

The structure of gellan in dilute aqueous solution.

A full assignment of high-field nmr spectra of gellan was obtained in dilute aqueous solution by performing a series of selective one-dimensional nmr experiments. The observed nuclear Overhauser effects (NOEs) cannot be interpreted assuming that each sugar residue is intrinsically rigid and in a chair conformation. In fact, the rhamnose residue gives strong NOE contacts coherent only with an equilibrium involving both a chair as well as a boat (or a hemiboat) conformation. Molecular dynamic calculations performed on a heptamer with a central rhamnose support the above finding, and show a structure based on a very stiff single chain in which it is present a flipping of the rhamnose residue. At low temperatures (5-20 degrees C) in very dilute solutions (0.018 mg/mL) nmr spectra show a splitting of the resonance due to the methyl group of rhamnose residue, thus confirming the presence of a slow equilibrium among different conformers.

Synthesis and preliminary characterisation of charged derivatives and hydrogels from scleroglucan.

The synthesis of negatively and positively charged polyelectrolytes from scleroglucan is described. Polycarboxylates were synthesised through nucleophilic substitution with chloroacetic acid or through a selective 2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPO)-mediated oxidation of the primary alcohol groups. Amine groups were introduced through nucleophilic substitution with 2-chloroethylamine or 3-chloropropylamine. Reaction conditions were varied to obtain insight into the influence of variables on the degree of substitution. The conformational behaviour of the obtained polyelectrolytes was studied as a function of pH, temperature and solvent. For the products with a low degree of modification, evidence of an ordered conformation was found, whereas the polymers with a higher degree of modification behaved as random coils in solution. The negatively charged polymers were reticulated using the Ugi four-component condensation, obtaining negatively charged hydrogels. The positively charged polymers were reticulated using diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dion, DES) to obtain positively charged hydrogels.

Comparative chemical evaluation of two commercially available derivatives of hyaluronic acid (hylaform from rooster combs and restylane from streptococcus) used for soft tissue augmentation.

Hyaluronic acid (HA) derivatives have been developed to try to enhance rheological properties of this molecule to make it suitable for various medical applications. The main dermatological application of HA derivatives is the augmentation of soft tissues, via injection into the dermis. HA derivatives are indicated for the correction of cutaneous contour deficiencies of the skin, particularly in cases of ageing or degenerative lesions or to increase lips. Two HA derivatives have been evaluated: Hylaform Viscoelastic Gel (Hylan B), derived from rooster combs and subjected to cross-linking, and Restylane, produced through bacterial fermentation (streptococci) and stabilized, as declared by the producer. In both cases the purpose is to improve HA theological characteristics and slow down its degradation once it is in contact with biological structures. Distribution of particle dimensions, pH, protein concentration and rheological properties have been investigated in order to evaluate their reliability as fillers for soft tissue augmentation. The results of the analyses showed that there are differences between Restylane and Hylaform. Especially as far as rheological characteristics are concerned, the results outline different structures of the products: Hylaform behaves as a strong hydrogel, Restylane as a weak hydrogel; rheologically Hylaform is clearly superior to Restylane. Hylaform contains a definitely minor quantity (about a quarter) of cross-linked hyaluronic acid than Restylane. Furthermore, although not declared by the manufacturer, Restylane contains protein, resulting from bacterial fermentation or added to enable cross-linking reaction; the quantity of proteins contained by Restylane can be as much as four times the quantity contained by Hylaform, for the same volume (1 ml). It is evident that Hylaform offers higher safety margin than Restylane. Furthermore, wide literature and 20 years of clinical experience on hyaluronan derived from rooster combs confirm the reliability of this derivative while we did not find evidence regarding about the safety of HA obtained from streptococcus.

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.

Synthesis and preliminary characterisation of new esters of the bacterial polysaccharide gellan.

Under the appropriate experimental conditions, ethyl, propyl, and methylprednisolon-21-yl esters of gellan can be obtained without significant degradation. At low degrees of esterification (de), depending on the ester moiety, the products are water-soluble, which allows the influence of hydrophilicity and charge density on their ability to assume an ordered conformation in dilute aqueous solution to be studied. With high de, the products were soluble only in organic solvents (e.g., methyl sulphoxide) with good film-forming capacity. The methylprednisolon-21-yl esters have been characterised in a preliminary manner in terms of drug-release kinetics.

Solution and gelling properties of polysaccharide polyelectrolytes.

Attention is focussed on the special class of polysaccharide polyelectrolytes belonging to the family of the microbial polysaccharides. But a few exceptions, these are high molecular weight water- soluble polycarboxylates with complex, regular structures. Complexity and regularity in primary structure--two features normally not shared by other polysaccharides of either vegetal or animal origin--quite naturally entail unique conformational propensities, special physico-chemical properties in bulk and in solution and, as a consequence, make these biopolymers of particular interest from both a basic research and an industrial standpoint. What is outlined in this presentation should demonstrate that for many exocellular microbial polycarboxylates the solution properties are, as expected, dictated by the conformation assumed by the polyelectrolytic chains which, in turn, are governed by several free energy terms in particular stemming from specific solvent-chain interactions, among which the coulombic contribution may play a minor role.

Structure and solution properties of tamarind-seed polysaccharide.

The major polysaccharide in tamarind seed is a galactoxyloglucan for which the ratios galactose:xylose:glucose are 1:2:25:2.8. A minor polysaccharide (2-3%) contains branched (1----5)-alpha-L-arabinofuranan and unbranched (1----4)-beta-D-galactopyranan features. Small-angle X-ray scattering experiments gave values for the cross-sectional radius of the polymer in aqueous solution that were typical of single-stranded molecules. Marked stiffness of the chain (C infinity 110) was deduced from static light-scattering studies and is ascribed partially to the restriction of the motion of the (1----4)-beta-D-glucan backbone by its extensive (approximately 80%) glycosylation. The rigidity of the polymer caused significant draining effects which heavily influenced the hydrodynamic behaviour. The dependence of "zero-shear" viscosity on concentration was used to characterise "dilute" and "semi-dilute" concentration regimes. The marked dependence on concentration in the "semi-dilute" region was similar to that for other stiff neutral polysaccharide systems, ascribed to "hyper-entanglements", and it is suggested that these may have arisen through a tenuous alignment of stiffened chains.

Influence of charged groups on the conformational stability of succinoglycan in dilute aqueous solution.

Viscosity, optical activity, and differential scanning calorimetry data clearly point out that partial and/or total removal of charged substituent groups, i.e. succinate and pyruvyl residues, from succinoglycan lead to water soluble derivatives exhibiting a higher stability order----disorder conformational changes with respect to the native polysaccharide. The new succinoglycan derivatives also exhibit very little, if any, hysteresis upon 'renaturation' (cooling) as opposed to the case of the parent polymer. The absence of ionized groups is thus beneficial, thermodynamically and kinetically, to the attainment in dilute aqueous solution of an ordered conformation by the uncharged succinoglycan backbone, as allowed by the regular enchainment of its constituent sugar residues.