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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. Gene delivery related research at UGent. PolExGene meeting Tuebingen, 20/12/2006. P. Dubruel , V. Vermeersch and E. Schacht. Introduction. Current status:

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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 Gene delivery related research at UGent PolExGene meeting Tuebingen, 20/12/2006 P. Dubruel, V. Vermeersch and E. Schacht

  2. Introduction Current status:  PEI and its derivatives show the highest transfection potential ‘Proton sponge’ theory of Behr:  pH lowers from 7.4 to 5-5.5  amount of protonated amines increases  influx of H+, Cl- and H2O  swelling of the endosomes  burst of the endosomes Behr J.P. Chimia (1997) 51, 34-36. Focus of our work: Synthesis and evaluation of polymers that mimmick the buffering properties of PEI, while showing a lower toxicity and having transfection ability

  3. Monomer & polymer synthesis Selected polymer: polymethacrylates Selected monomers: amine functions with varying pKa  tertiary amine, pyridine group and imidazole group other functional groups  carboxylic acid pKa 10 5 6 5

  4. Monomer & polymer synthesis + comonomer + AIBN PDMAEMA DMAEMA P(DMAEMAx-co-MAy) P(DMAEMAx-co-HYMIMMAy) P(DMAEMAx-co-HENIMAy)

  5. Monomer & polymer synthesis Molecular weight determination: gel permeation chromatography or static light scattering

  6. Physico-chemical evaluation EtBr exclusion test: - based on the exclusion of EtBr from the DNA minor groove - ex = 510 nm and em = 590 nm PDMAEMA P(DMAEMAx-co-HYMIMMAy) All polymers are able to condense DNA Mw and the chemical composition affect on the DNA condensation process

  7. P1 P2 P3 P4 DMAEMA P5 P6 P7 P8 HENIMA MA P9 P10 P11 HYMIMMA Physico-chemical evaluation Agarose gel electrophoresis: separation of condensed and free DNA by V

  8. Biological evaluation Transfection studies:  cell lines: - human retinal pigment epithelial cells (D407 cells) - kidney cells of the African green monkey (Cos-1 cells)  DNA: - plasmid DNA encoding for the enzyme -galactosidase (pCMV) - determination of -gal activity or mass  Cos-1 cells: PDMAEMA and P(DMAEMA0.9-co-HENIMA0.1) Polymethacrylates possess a lower transfection efficiency and toxicity compared to PEI

  9. Biological evaluation Confocal microscopy studies: Cos-1 cells double labeling experiments polymer label: Oregon GreenTM endosome label : Lysotracker Red red signal endosomes without complexes green signal polyplexes that are not located in the endosomes yellow signal polyplexes that are located in the endosomes

  10. Biological evaluation 90 minutes 15 minutes 45 minutes PEI 15 minutes 30 minutes 240 minutes DMAEMA based polymers

  11. Biological evaluation Synthesis of modified hyaluronic acid PEI-DNA complexes PDMAEMA-DNA complexes Study of polyplex-glycoaminoglycan interaction

  12. Application of Penetratin for gene delivery •  Combination of polyplexes and Penetratin:  Penetratin: - 16 amino acid cationic peptide (arginine and lysine) • - corresponds to residues 43-58 of the 3rd helix of the • homeodomain of the Antennapedia homeoprotein (a Drosophila • transcription factor) • - cell uptake does not occur via endocytosis (> < complexes) but • through membrane translocation •  Effect on transfection potential, cell toxicity and cell uptake of complexes •  Penetratin addition (up to 100 µM) to pre-formed polymer-DNA complexes

  13. Biological evaluation Transfection study: Cos-1 cells P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.1) P(DMAEMA0.87-co-AEMA0.13) 2/1 charge ratio 4/1 charge ratio Conclusions: - Penetratin increases the transfection efficiency of polymethacrylates - Penetratin does not alter the toxicity of polymethacrylate-DNA complexes

  14. Biological evaluation 0.5 h 1 h 4 h no Penetratin 25 µM Penetratin

  15. 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)

  16. 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)

  17. 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 labeled

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

  19. 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)

  20. 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

  21. 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)

  22. 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)

  23. 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

  24. 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)

  25. 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

  26. 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

  27. 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,…

  28. Thank you! Questions?

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