Stefan Vansteenkiste

Protein Adhesion at Poly(ethylene glycol) Modified Surfaces

The inhibition of protein adsorption to the surfaces of biomedical devices is a crucial requirement for avoiding implant-associated infections or thrombus formation on blood-contacting artificial surfaces and thus for increasing the long-term biocompatibility of the devices. Here, the use of surface plasmon resonance and scanning force microscopy using protein-modified tips (see figure) to study protein adhesion on poly(ethylene glycol) (PEG) grafted polymer materials is discussed. The PEG-rafted materials are revealed to have significantly reduced affinity to proteins.

Fate of glycosylated dextrans after in vivo administration

Abstract Polymers bearing galactose or mannose residues accumulate in the liver after intravenous administration to rat or mice. In this study dextrans carrying d -galactose residues were injected intravenously in rat. Their rates of blood clearance and body distribution were measured. d -Galactosylated polymers were cleared more rapidly in comparison with non-glycosylated control polymer. The former are more easily captured by the liver. Dextran substituted with tri- d -galactose units was cleared faster than mono d -galactose substituted dextran. Introduction of FITC groups in dextran or d -galactosylated dextran (D.S.: 0.7%) resulted in a faster internalization by the liver. The effect of the galactose co-substituents was overridden by that of the FITC-moieties. d -Mannosylated dextran was evaluated as a blocker of liver d -mannose receptors. It was shown that co-administration of d -mannosylated dextran to i.v. administered ricin A immunotoxin significantly decreased the rate of blood clearance and liver uptake of the immunotoxin. Body distribution experiments carried out at 24 h indicated the liver blocking to be transient.

Glucose oxidase as a tool to study in vivo the interaction of glycosylated polymers with the mannose receptor of macrophages

Abstract In the present paper, we report the competition for the mannose-specific lectin of mononuclear phagocytes between two potential drug carriers, namely dextran and poly-α,β-[N(2-hydroxyethyl)-D,L-aspartamide] (p-HEA) both modified by either α-D-mannose or β-L-fucose residues, and glucose oxidase (G.O.) following intravenous coinjection into mice. Native dextran or p-HEA did not influence the plasma half-life time of G.O. On the other hand, coinjection of an excess of either α-D-mannosylated or β-L-fucosylated dextran of comparable sugar content did increase the circulation half-life time significantly. The extent by which the T1/2 of G.O. was prolonged, depended on sugar loading and the amount of competing polymer. Comparison between β-L-fucosylated and α-D-mannosylated dextran revealed a slightly more efficient receptor inhibition by mannose. The effect of the macromolecular carrier nature was clearly demonstrated by comparison between dextran and p-HEA conjugates. All glycosylated p-HEA derivatives retarded the blood clearance of G.O. less than the dextran analogues. Further competition experiments revealed a rather peculiar in vivo behaviour of modified dextrans, probably due to adsorption phenomena on blood cell membranes.

Synthesis of Glycosylated Dextrans

Dextran fractions with a narrow molecular weight distribution were modified using the 4-nitrophenyl chloroformate activation method. The activated polymers were subsequently reacted with a small amount of L- tyrosinamide and a number of selected peracetylated ω-amino glycosides of D- mannose, D-galactose, L-fucose and L-rhamnose and including some cluster de rivatives. All monosaccharides were linked to the polymer chain via a carbon C-6 spacer. The number of L-tyrosinamide units introduced could be accurately deter mined by UV absorption spectroscopy. On the other hand, the degree of substi tution by a glycoside ligand was calculated from 1H NMR data.

Impact of hydrated lime on the durability of SMA mixtures: laboratory and field evaluation

The work presented in this paper describes the research activities of the task group ‘Properties of filler aggregates’ with respect to the durability of Stone Mastic Asphalt (SMA) mixtures, which was conducted recently at the Belgian Road Research Centre. The main objective of this study comprises the effect of hydrated lime on the durability of SMA mixtures. The methodology developed in this study includes in an initial phase the design in the laboratory of a ‘critical’ SMA10 mixture. Such a ‘critical’ SMA10 mixture was considered to be characterised by a high water sensitivity and, therefore, a low durability. During this process, attention was paid to both the SMA composition as well as to the compaction energy applied in order to provide suitable test samples. Subsequently, by making use of such a ‘critical’ SMA10 mixture, the effect of the filler nature on the durability of the SMA mixtures was established; in particular both the modification by as well as the optimal content of hydrated lime was studied. Additionally, the possible effect of hydrated lime on the workability of latter SMA mixtures was investigated. Finally, the results obtained in the laboratory were complemented by a field study and a monitoring campaign. This survey comprises the evaluation of the impact of hydrated lime on the compactibility and the water sensitivity on test sections as well as a yearly follow up of the performance of a series of SMA10 mixtures varying in the type of filler material used.

Phosphonylation of purified human, canine and porcine cholinesterase by soman: Stereoselective aspects

Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400-600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+)P(+/-)-soman and C(-)P(+/-)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(-)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(-)-epimers. Individual soman isomers were isolated from C(+)- and C(-)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+)P(-)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(-)P(-), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(-)-isomers at a slightly higher rate than the human enzyme.

In vitro binding specificity of glycosylated dextrans to the asialoglycoprotein receptor of primary hepatocytes

Abstract The binding of dextran molecules bearing single (6.0 mol%) or clusters of three (8.5 mol%) 1-O-linked galactose residues to primary rat hepatocytes has been characterised. Hepatocyte-accumulation of radiolabelled monoantennary galactosylated dextran (10 ng/ml) was down-regulated with increasing time in culture, falling from a rate of 5.6 (ng/mg cell protein)/h (freshly isolated cells) to 4.1 (ng/mg cell protein)/h and 1.2 (ng/mg cell protein)/h after 24 h and 48 h, respectively. Incubation in dinitrophenol (100 μg/ml) or low temperature (4°C) decreased accumulation by 72% and 55%, respectively, suggesting inhibition of an active endocytic internalisation. Control (unmodified) and mannosylated dextrans showed negligible cell association. Accumulation of trisantennary galactosylated dextran (6.1 ± 1.2 (ng/mg protein)/h) was greater than that of the monoantennary form (4.5 ± 0.9 (ng /mg protein)/h), reflecting greater hepatic capture of the former in vivo. Free monosaccharides ( d -galactose, d -mannose, l -mannose, d -galactosamine and l -rhamnose) showed no inhibition (up to 5 mM) of hepatocyte-binding of either galactosylated dextran. The monoantennary galactosylated and trisantennary galactosylated forms showed mutual competition for binding (IC 50 values for mutual antagonism of 16.4 and 0.9 μM, respectively, measured as galactose content), indicating recognition by the same receptor with stronger binding of the trisantennary form.

Effect of Extraction and Recovery Procedure on the Determination of PmB Content and on the Properties of the Recovered Binder

ABSTRACT In Belgium, the commonly used test method for the determination of the binder content of a bituminous mixture is based on the extraction of the soluble bitumen with a centrifuge. This step is followed by a recovery of the solvent with a rotary evaporator when the binder is yet to be characterized. The procedures for pure binders are clearly described in the European standards, but this is not the case for polymer modified bitumens (PmBs). Consequently, a research project has been launched to evaluate, in the case of bituminous mixtures with PmB both the effect of extraction on the binder content as well as the effect of extraction and recovery on the properties of the recovered PmB. In this study, the use of several solvents and dissolution methods has been compared.

Coloured Asphalt Pavements: Mix Design and Laboratory Performance Testing

Coloured asphalt pavements are increasingly used in large public areas and at dangerous intersections such as crossroads, roundabouts or pedestrian crossings. In the former application, the role of the coloured pavement is often to give the space a particular aesthetic character or to integrate it well in its surroundings; in the latter, its major role is to enhance the safety of users by improving visibility and road legibility. However, coloured pavements are also subjected to traffic and climate-induced stresses and must, therefore, exhibit a similar mechanical performance as their uncoloured counterparts. Desired colours are obtained by using specific materials such as coloured aggregates, pigments and clear binders. However, the application of latter constituents may affect the performance and durability of coloured asphalt mixtures. Therefore, in a first step, BRRC (Belgian Road Research Centre) determined the characteristics of these particular materials such as the rheological behaviour of clear binders both at high and low temperatures and the stiffening effect of pigments acting as filler. Subsequently, the impact of the material characteristics on volumetric mix design was investigated. In a next phase their effect on the mechanical performance of coloured mixtures was studied in the laboratory. The latter performance was evaluated by testing for water sensitivity, rutting resistance and low-temperature cracking. This contribution summarizes the major findings from the study.

Influence of hydrated lime on the field performance of SMA10 mixtures containing polymer modified binder

In literature, the beneficial effect of hydrated lime on the durability of asphalt mixtures has been demonstrated extensively, especially while performing laboratory testing. However, experimental evidence on the impact of hydrated lime in combination with the use of polymer modified bitumen (PmB) on the field performance of an asphalt pavement is rather scarce. Therefore, an initiative was taken to set up a test program of which the main objective was to gain as much as possible experience from the field.Hence, the comparative study focused on the assessment of the performance of SMA10 variants by the construction of test sections in combination with a series of laboratory tests. The impact of hydrated lime on the performance of SMA10 variants was investigated at different stages. Prior and during the construction of the test sections, the effect of hydrated lime on the workability was monitored by carrying out either gyratory compaction tests in the laboratory or by a follow up of the in situ compaction by making use of the -nuclear gauge. Following construction, appropriate test specimens were taken by coring in order to probe for the resistance to rutting, the water sensitivity and finally the resistance to ravelling. This paper discusses and draws conclusions from this comparative field study with respect to the use of hydrated lime in SMA10 mixtures containing a polymer modified bitumen.