Promotion de l'adhésion cellulaire par le couplage covalent de peptidomimétiques sur une membrane polymère

Notre recherche a pour objectif la biocompatibilisation active de polymères de synthèse. La stratégie utilisée consiste à greffer de manière covalente en surface du polymère des signaux synthétiques. Parmi les nombreuses applications de cette stratégie, nous nous sommes intéressés à la promotion de l'adhésion cellulaire sur le poly(éthylènetéréphtalate) (PET). Ce matériau possède des propriétés déjà amplement exploitées dans des application biomédicales. Durant cette thèse, nous avons abordé plusieurs aspects dans le contrôle et l'évaluation du substrat modifié. Afin de réaliser un greffage efficace, nous avons étudié les sites réactifs du PET (fonctions acides vs fonctions alcools) ainsi que les méthodes de couplage via la chimie organique appliquées à l'interface solide-liquide (surface wet chemistry). En relation avec cette étude, nous avons synthétisé et évalué différents bras d'espacements afin d'établir les plus performants lors d'un greffage en surface. L'adhésion cellulaire est un phénomène s'établissant via des récepteurs cellulaires. Par le greffage de motifs de reconnaissance de ces récepteurs, nous envisageons d'accroître le taux d'adhésion cellulaire. Pour cela, nous avons réalisé le design et la synthèse de peptidomimétiques de la séquence RGD, motif universellement reconnu par les intégrines. Ces dernières sont une des grandes familles de récepteurs cellulaires, très étudiés en chimie médicinale. Finalement, notre évaluation biologique se scinde en 2 parties. D'une part, greffés en surface, nos peptidomimétiques ont pour effet recherché, la promotion de l'adhésion cellulaire. D'autre part, la saturation des récepteurs cellulaires par les ligands en solution inhibe l'adhésion cellulaire via ces récepteurs. Ce dernier point est un aspect développé par de nombreux groupes pharmaceutiques dans le traitement de certaines maladies. Un test plus poussé de binding moléculaire a pu être effectué nous apportant des informations quand au phénomène de reconnaissance. A partir des premiers résultats obtenus, nous avons pu optimiser le design selon un protocole itératif basé sur une analyse théorique par modélisation plus poussée.(CHIM 3)--UCL, 200

Cell adhesive PET membranes by surface grafting of RGD peptidomimetics.

A non-peptide mimic of the Arg-Gly Asp (RGD) active sequence of adhesive proteins (such as vitronectin) has been equipped with two different spacer-arms for surface anchorage. The covalent grafting on poly(ethylene terephthalate) (PET) membrane was realized via the activation of the hydroxyl polymer chain-ends by tosylation followed by nucleophilic substitution. The surface density of peptidomimetics was determined by X-ray photoelectron spectroscopy (XPS), on the basis of F/C atomic ratios since a fluorine tag was incorporated into the RGD-like compounds. The biological activity of soluble peptidomimetics was evaluated versus isolated human integrin alpha(v)beta(3) (vitronectin receptor), and versus CaCo2 cells. Inhibition of cellular adhesion was observed after pre-incubation of CaCo2 cells with soluble peptidomimetics. On the other hand a significant promotion of cellular adhesion resulted from the surface grafting of peptidomimetics on the PET culture substrate. The best performance was obtained with the RGD-like integrin ligand bearing a triethylene glycol spacer-arm

Chemoselective O-methylation of N-acylated/sulfonylated tyrosine derivatives

Methyl ethers (6a and 6b) of N-trifluoroacetyl- and N-(m-trifluoromethyl) phenylsulfonyl-6-nitro-tyrosine t-butyl ester were readily prepared by modified Mitsunobu reaction (DPPE. DIAD, MeOH). Williamson (Mel, K2CO3 or LiCO3 or NaOH under phase transfer) and classical Mitsunobu conditions (PPh3, DEAD, MeOH) gave O,N-dimethylated derivatives (7a and 7b) as side or main products. O- versus N-selectivity in tyrosine methylation reactions depends on both pK(a) values and steric factors. (C) 2002 Elsevier Science Ltd. All rights reserved

Effect of the chemical nature and length of spacer arms on the covalent grafting of fluorinated molecular probes at the surface of poly(ethylene terephthalate) membrane

A series of fluorinated molecular probes were synthesized that are characterized by spacer arms of various lengths and polarities. Previous molecules (1, 2a, 2b, 2c, 3a, 3b) were covalently fixed on the surface of poly(ethlylene terephthalate) (PET) membranes via activated hydroxyl chain endings. X-ray photoelectron microscopic analysis of the grafted samples allowed us to quantify the PET surface reactivity; the results were within 40-50 pmol/cm(2) of fixed probes, independent of the length and hydrophilicity of the spacer arms. (C) 2002 Wiley Periodicals, Inc

An Unprecedented Surface Oxidation of Polystyrene Substrates by Wet Chemistry under Basic Conditions.

The surface of polystyrene substrates has been modified by wet chemistry consisting of a treatment with sodium hydroxide in a water-methanol solution at 50 degrees C for 15 h, under air atmosphere. The resulting samples were analyzed by XPS and AFM. The surface functional groups (hydroxyl and carboxyl functions) were assayed by radiolabeling. All the results are consistent with a surface oxidation process

Radiolabeling of Pluronic amphiphilic copolymer for adsorption studies.

A new pathway for the radiolabeling of Pluronic PE6800 was developed. In a first step, the CH(2)-OH end groups of the copolymer were substituted by tosylates; in a second step these were reduced by [3H]-NaBH(4) to obtain tritiated chain ends. The final product was shown to be a mixture of native, tosylated, and reduced Pluronic containing 1 tritium atom per 1110 Pluronic molecules. The labeling procedure did not affect the molecular weight distribution nor the adsorption isotherm of the copolymer on polystyrene plates. A plateau value of about 0.7 microg/cm(2) is reached at a concentration in solution of 500 microg/ml, i.e., much lower than the cmc. Upon drying, the Pluronic adsorbed layer reorganizes in particles with a size of about 30 to 60 nm which cover about 15% of the substratum surface. This observation is of great importance for the design of protein-resistant surfaces by adsorption of Pluronic

Novel RGD-like molecules based on the tyrosine template design, synthesis, and biological evaluation on isolated integrins alpha(V)beta/alpha(IIb)beta(3) and in cellular adhesion tests

RGD (Arg-Gly-Asp) peptidomimetics have been designed for covalent anchorage on biomaterials. The tyrosine template was thus equipped with (i) a basic side chain of various flexibility, (ii) an acidic side chain, which incorporated the XPS fluorine tag, and (iii) a spacer-arm terminated by a primary amine for surface grafting. The most active compounds showed IC50 values in the nanomolar range versus isolated human integrins alpha(v)beta(3) and alpha(IIb)beta(3). Preincubation of CaCo2 cells with soluble peptidomimetics (2 and 19a) prevented cellular adhesion on culture plates coated with vitronectin. On the other hand, peptidomimetics (19a and 19b) immobilized on a poly(ethylene)terephthalate membrane (PET) promoted CaCo2 cells adhesion. A modeling study at the ab initio level in MINI-1' basis allowed to compare the various synthetic ligands of integrins and to propose novel pharmacophore structures. (C) 2004 Elsevier Ltd. All rights reserved

Submicrometer-scale heterogeneous surfaces by PS-PMMA demixing

Patterned surfaces were created using two polymers: polystyrene (PS) on the one hand, and either poly(methyl methacrylate) (PMMA) or poly(methyl methacrylate)-poly(methacrylic acid) (PMMA-PMAA) on the other hand. PMMA was dissolved in a solvent of PS; this solution was then spin-coated on a PS support that partially dissolved during the process. The materials were analyzed by water contact angle measurement, XPS, ToF-SIMS and AFM. The effect of the solvent on the final surface morphology was strongly marked. With chloroform, the acrylic polymer was the major surface constituent, possibly because of the high evaporation rate of this solvent. With toluene, which is a better solvent for PS compared to PMMA, the obtained surface was almost exclusively constituted of PS. The use of chlorobenzene provided inclusions of acrylic polymer in PS, both polymers being exposed at the outermost surface. The surface morphology presented rings, the interior of which consisted of the acrylic polymer, while the rest of the surface was made of PS. (C) 2004 Elsevier Ltd. All rights reserved