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Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health

PolExGene. Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health. UGhent contribution to PolExGene . PolExGene kick-off meeting Ghent, 23-24/08/2006. Dr. P. Dubruel and Prof. E. Schacht. Summary UGhent tasks

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Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health

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  1. PolExGene Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health UGhent contribution to PolExGene PolExGene kick-off meeting Ghent, 23-24/08/2006 Dr. P. Dubruel and Prof. E. Schacht

  2. Summary UGhent tasks Development of CPP-containing polymers (WP2) Development of CIP-containing polymer membranes by solvent casting and electrospinning (WP3) Preparation of plasmids and CPP-containing polyplexes(WP4)

  3. DNA binding groups CPP WP 2 Development of CPP-containing polymers WP 2.1 Synthesis and characterisation of cationic polymers and peptides  biodegradable poly--amino acids •Synthesis: R2 = DNA binding groups groups,… e.g. amine, agmatine,... Copolymers with arginine will be prepared in the same way •Characterisation:structural analysis (1H-NMR, IR and UV) molecular weight analysis (GPC, light scattering)

  4. CPP WP 2.2 Conjugation of CPP to cationic polymers  cysteine/maleimide coupling chemistry (1)  reactive ester coupling chemistry (2) Selected variables: polymer side group (R2) and molecular weight structure and density of CPP spacer between polymer and CPP combination of different CPP Selected number of polymer derivatives will be fluorescently labelled

  5. WP 2.3 Synthesis and characterisation of Au binding (bio)polymers e.g. functionalised hyaluronic acid, GAGs,... •Synthesis: •Application: Work Package 4

  6. Summary UGhent tasks Development of CPP-containing polymers (WP2) Development of CIP-containing polymer membranes by solvent casting and electrospinning (WP3) Preparation of plasmids and CPP-containing polyplexes(WP4)

  7. WP 3 Development of CIP-containing polymer membranes by solvent casting and electrospinning • Membrane function: support the attachment and growth of transfected cells • Material selection: crosslinkable semi-synthetic polymers crosslinkable gelatin crosslinkable esterified hyaluronic acid crosslinkable chondroitin sulphate chondroitin sulphate (R1 = H or SO3H, R2 = H or SO3H) partially esterified hyaluronic acid

  8. WP 3 Development of CIP-containing polymer membranes by solvent casting and electrospinning • Membrane function: support the attachment and growth of transfected cells • Material selection: crosslinkable semi-synthetic polymers crosslinkable gelatin crosslinkable esterified hyaluronic acid crosslinkable chondroitin sulphate crosslinkable gelatin (GEL) crosslinkable chondroitin sulphate (CS)

  9. WP 3 Development of CIP-containing polymer membranes by solvent casting and electrospinning • Membrane preparation: solvent casting and/or electrospinning Solvent casting: casting of methacrylate/methacrylamide modified polymers photo-initiator + irradiation (visible light)  crosslinking  polymer films or porous polymer membranes (via cryogenic treatment) Electrospinning: production of membranes composed of non-woven nanometer polymer fibres used alone/combined with solvent casting Coupling of CIP: prior to membrane preparation Cardiovascular application: direct polymer coating on vascular grafts • Membrane characterisation: AFM, SEM (surface roughness) ATR-IR, XPS, TOF-SIMS (surface composition)

  10. http://www.che.vt.edu Solvent casting: h plastic spacer glass plates Electrospinning: Apply V between reservoir and collector plate V > Vcr Fr >  formation of an electrically charged polymer jet

  11. Summary UGhent tasks Development of CPP-containing polymers (WP2) Development of CIP-containing polymer membranes by solvent casting and electrospinning (WP3) Preparation of plasmids and CPP-containing polyplexes(WP4)

  12. WP4 Preparation of plasmids and CPP-containing polyplexes WP 4.2 Methods for monitoring polyplex formation • Aim: study DNA condensing ability of cationic polymers • Methodology: 1. fluorescence/UV-based techniques: EtBr exclusion tests agarose gel electrophoresis 2. surface plasmon resonance: 3. light scattering-based techniques: dynamic light scattering (size) zeta potential measurements (charge) • Feed-back: apply monitoring techniques for polymer/polyplex optimisation

  13. WP 4.3 Comparison of CPP immobilisation strategies • Aim: comparison of CPP immobilisation methods • Strategy: 1. covalent coupling before polyplex formation 2. covalent coupling after polyplex formation 3. physical mixing of CPP and pre-formed polyplexes • Analysis: DLS, -potential, EtBr, electrophoresis, SPR,... WP 4.4 Functionalisation of polymer membranes with polyplexes

  14. WP 4.3 Comparison of CPP immobilisation strategies • Aim: comparison of CPP immobilisation methods • Strategy: 1. covalent coupling before polyplex formation 2. covalent coupling after polyplex formation 3. physical mixing of CPP and pre-formed polyplexes • Analysis: DLS, -potential, EtBr, electrophoresis, SPR,... WP 4.4 Functionalisation of polymer membranes with polyplexes • Strategy: anionic membranes: dipcoating cationic membranes: pre-coating with proteins • Analysis: XPS, TOF-SIMS, AFM, SEM,…

  15. Thank you! Questions?

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