Vittorio Crescenzi

Gellan-based systems for ophthalmic sustained delivery of methylprednisolone

Abstract Gellan is an anionic exocellular polysaccharide of microbial origin, having a characteristic property of cation-induced gellation. In the current study, gellan was evaluated for applications in ocular sustained release devices. A methylprednisolone (MP) ester of gellan (gellan-MP) was synthesized. Sustained release dosage forms evaluated were gellan-MP films, gellan films with physically incorporated MP and eye drops of MP suspended in a 0.6% w/w/ gellan dispersion in water. The control dosage form was a suspension of MP in normal saline. In vitro release ofMP from the test dosage forms was determined in a pH 7.4 phosphate buffer at 32 °C using a rotating bottle apparatus. MP concentrations in the tear fluid of New Zealand white rabbits were measured after ocular application of the dosage forms. In vitro, the gellan-MP films released covalently bound MP in an approximate zero-order pattern, whereas the release of physically incorporated MP from the gellan eye drops and films followed a square root of time relationship and anomalous kinetics, respectively. Compared with the MP suspension control, the gellan-MP films yielded an approximately 4-fold higher area under tear fluid concentration vs time curve, AUC0–8h, but exhibited a tendency to slip out of the eye due to a high degree of swelling. The in vivo release from films containing physically incorporated MP showed higher variability and provided mean AUC0–8h values approximately equal to the control values. The gellan eye drops containing MP yielded 2.6-fold higher AUC0–8h values than the control and also provided ease of administration. Gellan solutions might thus provide a versatile vehicle for ocular sustained release of drugs. The results also show that the gellan-MP ester can be used to increase the residence time of methylprednisolone in the tear fluid of rabbits.

The influence of side-chains on the dilute-solution properties of three structurally related, bacterial anionic polysaccharides

Abstract Viscometric, calorimetric, and chiroptical data collected by using dilute aqueous solutions of the extracellular polysaccharides [Me4N+ salt form] from Pseudomonas elodea (S-60), Alcaligenes (ATCC 31555) (S-130), and Alcaligenes (ATCC 31961) (S-194) over a range of ionic strength (Me4NCl concentrations) and temperature are reported. The data demonstrate that increasing the ionic strength of dilute solutions of the nonbranched S-60 at 25° promotes a distinctly co-operative, conformational transition over a narrow range of added salt concentration. Moreover, over the range of ionic strength examined, S-60 also undergoes a thermally reversible, conformational transition which exhibits no hysteresis: the temperatures at the midpoint of the sigmoidal transitions (TM) increase moderately with increasing ionic strength. This conformational ordering has been monitored under conditions where S-60 does not gel. However, because both higher concentrations of S-60 at the same ionic strength, and higher ionic strengths at the same S-60 concentration, lead to gel formation, it is likely that the conformational ordering we have observed is the one involved in the gelation of S-60. The data also indicate that the two different modes of branching along the S-60 backbone present in S-130 (α- l -rhamnose and α- l -mannose linked to O-3 of the d -glucose after the d -glucuronic acid in the tetrasaccharide repeating-unit) and S-194 [β- d -glucopyranosyl-(1 → 6)-α- d -glucopyranosyl units linked to O-6 of the d -glucosyl residue before the d -glucosyluronic acid] mediate against conformational ordering. This is consistent with the fact that S-60 forms firm aqueous gels, whereas S-130 and S-194 are non-gel-forming, but give stable, highly viscous solutions. In addition, the measurements indicate that the carboxyl group in S-30 is in a screened, molecular environment. It is suggested that this screening could arise from hydrogen bonding involving the l -rhamnose- l -mannose side-chains.

Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC-diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 microg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.

Comparative analysis of the behavior of gellan gum (S-60) and welan gum (S-130) in dilute aqueous solution☆

Abstract Optical rotation, circular dichroism, and microcalorimetric data clearly and consistently show that gellan gum, S-60 (Me 4 N + form), undergoes in water at 25° a rather sharp conformational transition upon increasing the concentration of added Me 4 NCl. Similar data show that S-60 behaves anomalously upon addition of Ca 2+ ions with, eventually, formation of aggregates and/or gels. The Me 4 NCl-induced conformational change of S-60 is thermally reversible with no hysteresis. In contrast, with welan gum, S-130 (Me 4 N + form), no evidence could be found for a dependence of chain conformation of the main external variables considered. Comparison of the circular-dichroism spectra of the two polysaccharides suggests that S-130 in water might be present in a stiff conformation similar to that assumed by S-60 in aqueous Me 4 NCl.

Atom Transfer Radical Polymerization as a Tool for Surface Functionalization

Atom transfer radical polymerization (ATRP) is receiving increasing attention as a method for surface functionalization. In this paper we discuss a simple way, based on the hydrophilicity of the substrate, for discriminating between surface and bulk polymerization in materials having a homogeneous distribution of initiator groups. This concept could allow a consistent simplification of the processing techniques and is therefore of high significance for the applicability of ATRP.

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.

The hydrophobic effect in aqueous solutions of nonelectrolytes. I. Self-interactions of alkylureas

In order to clarify some aspects of the hydrophobic interactions, the enthalpies of dilution of monoethylurea, 1,3-dimethylurea, and 1,3-diethylurea have been determined calorimetrically at 25°C. The calorimetric data, expressed in terms of excess enthalpy, permit the evaluation of the pair and triplet interaction coefficients. The analyses of these and of the analogous coefficientsgxx andgxxx, derived from osmotic data, indicate a driving force favorable to the interactions among the hydrated solute molecules. Nevertheless, the positive values of thehxx andhxxx coefficients seem to suggest that the source of the effect is a rearrangement of the water molecules rather than a direct association of the solute molecules. There are evidences of a strict correlation between the enthalpic and the entropic effects.

NMR study of a novel chitosan-based hydrogel

Abstract Chemical gels are obtained by reacting chitosan with 1,1,3,3-tetramethoxypropane (TMP, a “masked” dialdehyde) and then reducing the polymeric Schiff-base networks with an excess of cyanoborohydride (NaBH 3 CN). The gels have been characterized by means of three different 13 C magic angle spinning NMR techniques: cross-polarization, cross-polarization with a simultaneous phase inversion and single pulse excitation. In this way we obtained spectra containing sufficiently resolved information for structural analysis. A quantitative evaluation of the cross-linking degree of chitosan–TMP networks is thus attainable.

New hydrogels based on carbohydrate and on carbohydrate-synthetic polymer networks

Abstract Novel synthetic routes recently explored in our laboratories in order to obtain crosslinked polymeric samples, comprising either chitosan or gellan as polysaccharidic components, are outlined. The former networks, in which different crosslinking partners have been used (i.e. oxidized cyclodextrin and telechelic poly(vinylalcohol)), can bear fixed positive charges while the latter are anionic. Zwitterionic networks formed by the template polymerization of acrylic acid onto chitosan with concomitant crosslinking (bisacrylamide) have also been prepared. Procedures adopted are simple and, once optimized, may lead to biocompatible hydrogels, with easily tunable physical properties, of potential interest in the biomedical area.

Tamarind seed polysaccharide: preparation, characterisation and solution properties of carboxylated, sulphated and alkylaminated derivatives

Abstract A range of derivatives of tamarind seed polysaccharide has been prepared, characterised and selected solution properties examined. Following oxidation of terminal sidechain galactose residues with galactose oxidase, subsequent oxidation and reductive amination have been used to prepare a range of carboxylated and alkylaminated derivatives respectively. Sulphated derivatives have been prepared by reaction with a sulphur trioxide-pyridine complex in dimethylformamide. The nature and extent of substitution have been characterised by potentiometric titration, infrared and 1H and 13C NMR spectroscopy. From the dependence of intrinsic viscosity on ionic strength (Smidsrod & Haug, 1971), carboxylated and sulphated derivatives are found to have characteristically stiffened backbones as found previously for the native polysaccharide (Gidley et al., 1991). Binding of divalent cations to carboxylated derivatives is shown to be relatively weak, although polymer precipitation was noted in the presence of Pb2+. Alkylaminated polysaccharides show only a modest decrease in surface and interfacial tension compared with the native polysaccharide, although significant foam formation and stabilisation was found for a nonylaminated sample. Following enzymic depolymerisation, this material showed a marked decrease in surface and interfacial tension suggesting that interfacial activity in alkylaminated tamarind polysaccharide is only apparent under disruptive solution conditions. The results of 1H NMR line width and T1 measurements before and after depolymerisation suggests that this is due to solution viscosity rather than specific interaction effects.