Giancarlo Masci

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.

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.

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.

Lipase-supported synthesis of peptidic hydrogels

Self-assembling gelling peptides are increasingly being investigated as defined biomatrices for biomedical applications. Using an enzymatic reaction to convert a precursor into a hydrogelator one can control or modulate functions and responses of a hydrogel, depending on its preparation conditions. This work details the self-assembly, under physiological conditions, of amphiphilic building blocks consisting of tripeptides (Phe3) linked to fluorenylmethoxycarbonyl (Fmoc) obtained by using a lipase to link an Fmoc–Phe amino acid to the dipeptide diphenylalanine (Phe2). The viscoelastic properties of the tripeptides obtained were investigated. SEM and AFM images of Fmoc–peptides confirmed that they self-assemble to generate supramolecular aggregates driven by π–π stacking interactions of the Fmoc groups.

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.

Chitosan Nanogels by Template Chemical Cross-Linking in Polyion Complex Micelle Nanoreactors

Chitosan covalent nanogels cross-linked with genipin were prepared by template chemical cross-linking of chitosan in polyion complex micelle (PIC) nanoreactors. By using this method, we were able to prepare chitosan nanogels using only biocompatible materials without organic solvents. PIC were prepared by interaction between chitosan (X(n) = 23, 44, and 130) and block copolymer poly(ethylene oxide)-block-poly[sodium 2-(acrylamido)-2-methylpropanesulfonate] (PEO-b-PAMPS) synthesized by single-electron transfer-living radical polymerization (SET-LRP). PIC with small size (diameter about 50 nm) and low polydispersity were obtained up to 5 mg/mL. After cross-linking of chitosan with genipin, the nanoreactors were dissociated by adding NaCl. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, and TEM. The size of the smallest nanogels was about 50 nm in the swollen state and 20 nm in the dry state. The amount of genipin used during reticulation was an important parameter to modulate the size of the nanogels in solution.

Novel thermosensitive calcium alginate microspheres: Physico-chemical characterization and delivery properties

The system described in this paper was obtained by soaking calcium alginate (CaAlg) microspheres in a water solution of poly-[(3-acrylamidopropyl)-trimethylammonium chloride-b-N-isopropylacrylamide] [poly(AMPTMA-b-NIPAAM)], a new block co-polymer recently synthesized by atom transfer radical polymerization (ATRP). The block co-polymer is characterized by a lower critical solution temperature (LCST) of 41 degrees C in aqueous 0.1 M NaCl solution, and can be anchored on the CaAlg microspheres by means of polyion interactions. Polycations (permanently positively charged blocks) and polyanions (free alginate carboxylic groups) interact, leading to microspheres with thermosensitive properties. As an effect of interaction with the microspheres the LCST of the co-polymer is lowered to 36-38 degrees C. In this temperature range a colloidal water suspension of the microspheres collapses, forming macroscopic aggregates. The new system shows, at human body temperature, an improved ability to carry and deliver both hydrophobic and hydrophilic molecules in comparison with unmodified CaAlg microspheres. The release properties of the microspheres loaded with different model drugs can be appropriately modulated by the amount of the poly(AMPTMA-b-NIPAAM). Furthermore, the microspheres show the interesting capability of retaining the activity of a loaded enzyme (horseradish peroxidase), used as a model protein. The results obtained indicate that the proposed drug delivery system may be suitable for drug depot applications.

Hydrogels based on pullulan derivatives crosslinked via a “living” free-radical process

Partially methacrylated pullulan was crosslinked by means of conventional and living radical polymerisations using, in the latter case, the reversible addition- fragmentation chain-transfer technique (RAFT). Several hydrogels were synthesised using methacrylated pullulan samples with different degrees of substitution, at different conversions using the RAFT technique for the first time. Network formation was monitored by means of rheological measurements and the ensuing samples characterised by swelling experiments and morphological studies. Hydrogels prepared by the living radical polymerisation always swelled to a greater extent than conventional gels having the same double bond conversion. The difference was more pronounced at low conversion. Scanning electron micro- scopy also showed that the porosity of the gels could be effectively regulated with the living polymerisation.

Synthesis and characterization of thermoresponsive N-isopropylacrylamide/methacrylated pullulan hydrogels

Thermosensitive hydrogels were prepared by free radical polymerization starting from a methacrylated pullulan derivative (acting as the cross-linker) and using N-isopropylacrylamide (NIPAAM) as the monomer. Several hydrogels were obtained by changing the monomer to cross-linker ratio. A significant thermosensitivity was observed only when the molar amount of NIPAAM incorporated in the network was at least eight times higher that of methacrylate groups on pullulan. The hydrogel with high amount of NIPAAM deswells more than 80% after the T-jump. The lower critical solution temperature of thermosensitive hydrogels decreases with increasing amount of NIPAAM. The mechanical properties of the hydrogels are strongly affected by the percentage of incorporated NIPAAM and by the temperature.