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Branched poly(ethylene glycols) (PEG2) are nowadays widely used for protein and peptides bioconjugation, for their favourable properties (such as the ability to protect the protein surface in an 'umbrella like' fashion). The discovery that mPEG(2)-LysMetbeta AlaOEt lost one mPEG chain during standard base-catalysed ester hydrolysis conditions prompted us to investigate the hydrolytic stability of such systems and the mechanism involved in the PEG chain loss. A series of branched PEGs, substituted with different aminoacids and dipeptides, have been prepared to test the influence of steric hindrance, chain lengths, ramification and Lys-AA amide substitution on hydrolysis. Unexpected results reveal an anchimeric assistance of the Lys-AaA amide proton to the hydrolysis of the carbamoyl moiety joining mPEG to the alpha-amino group of lysine through the formation of an hydantoin system.

Within the present study various polycarbophil (PCP)-serine protease inhibitor conjugates were synthesized and the influence of different spacers on their inhibitory efficacy was evaluated in vitro. Results demonstrated that 4.2+/-0.15 units (n=3; +/-SD) of alpha-chymotrypsin were inhibited by 50% utilizing 0.86% (w/v) of a PCP-tetramethylenediamine (TMDA)-chymostatin 20:1 conjugate. In contrast, only 0.6+/-0.05 units (n=3; +/-SD) of alpha-chymotrypsin were inhibited by a corresponding PCP-poly(ethylene glycol) (PEG)-chymostatin conjugate. Inhibitory effects of PCP-TMDA-antipain and -elastatinal conjugates towards trypsin and elastase, respectively, were also significantly higher (P<0.05) than those of corresponding PCP-PEG-inhibitor conjugates. Hence, the great impact of the molecular size as well as the structure of the spacer on resulting polymer-inhibitor conjugates could be demonstrated. The small and rigid C4-spacer TMDA (molecular weight (MW) 161.1) was thereby shown to be highly advantageous over a long, hydrophilic and flexible PEG-diamine spacer (MW 3400). Results obtained should provide helpful basic knowledge for the development of mucoadhesive polymer-inhibitor conjugates used as auxiliary agents for the oral administration of peptide drugs.

The paper discusses general problems in using PEG for conjugation to high or low molecular weight molecules. Methods of binding PEG to different functional groups in macromolecules is reported together with their eventual limitations. Problems encountered in conjugation, such as the evaluation of the number of PEG chains bound, the localisation of the site of conjugation in polypeptides and the procedure to direct PEGylation to the desired site in the molecule are discussed. Finally, the paper reports on more specific methods regarding reversible PEGylation, cross-linking reagents with PEG arms, PEG for enzyme solubilization in organic solvent and new polymers as alternative to PEG.

Membranes or microcapsules made from polyphosphazenes bearing amino acid side groups are proposed for the treatment of periodontal diseases. Polyphosphazene membranes, prepared with alanine ethyl ester and imidazole in the molar ratio of 80:20 as phosphorus substituents, gave a degradation rate that corresponded to the healing of the bone defect. These membranes were much more successful in promoting healing of rabbit tibia defects than polytetrafluoroethylene membranes. Antibacterial or anti-inflammatory drugs, useful in periodontal tissue regeneration, could be entrapped in the polyphosphazene membranes and released both in vitro and in vivo at a rate that ensured therapeutic concentrations in the surrounding tissue. Polyphosphazene microspheres, prepared with phenylalanine ethyl ester as a phosphorus substituent and loaded with succinylsulphathiazole or naproxen, were also obtained. The kinetics of release from these matrices were very convenient in yielding local concentrations of the two drugs that are useful per se or when mixed with hydroxyapatite for better bone formation.

Nerve regeneration experiments were carried out using tubular nerve guides of poly[(ethylalanato)1.4(imidazolyl)0.6phosphazene] (PEIP). By means of in vivo tests, this polymer was found to be biodegradable and transformed into harmless products. The tubular nerve guides were prepared by deposition of the dissolved polymer on a glass capillary tube, followed by evaporation of the solvent (methylene dichloride). After transectioning, rat sciatic nerve stumps were immediately sutured into the ends of 10-mm-long polymer tubes. On removal of the prosthesis, after implantation for 45 d, a tissue cable was found bridging the nerve stumps in all cases. Histological analysis revealed that the tissue cable was essentially composed of a regenerated nerve fibre bundle. A parallel series of experiments was undertaken to compare the use of silicone tubes that are not biodegradable and are most frequently used for studies of nerve regeneration with tubulization techniques. The advantages of biodegradable PEIP tubular nerve guides used for peripheral nerve repair are discussed.

Different families of functionalized polymers with potential as biomaterials, or for biomaterial modification, have been investigated. In particular, degradation studies have been performed on poly(amidoamines), a family of polymers obtained by polyaddition of amines to bisacrylamides, and endowed with heparin-complexing ability. Some new poly(amidoamines) with more resistance towards hydrolytic degradation than traditional ones have been discovered. Other ter-amino polymers deriving from the polyaddition of ter-amino functionalized bis-thiols to bis-acrylic esters, or other activated unsaturated compounds, have been studied. Their quaternarization products have been proven, in a parallel work, to act as powerful antimicrobial agents. By performing in situ the polyaddition reaction, semi-interpenetrated networks based on silicone rubber and the same polymers have been prepared. Finally, end-functionalized amphiphilic oligomers have been prepared by radical polymerization techniques, and their use for enzyme modification considered.