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UGent contribution to PolExGene (WP 2, 3 & WP 4)

U. PBM. - G ent. Polymer Chemistry & Biomaterials Group. UGent contribution to PolExGene (WP 2, 3 & WP 4). PolExGene Technical Meeting Tübingen 18-19/05/2009. PolExGene. V.Vermeersch , S. Van Vlierberghe, V. Toncheva P. Dubruel. Workpackage Contents.

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UGent contribution to PolExGene (WP 2, 3 & WP 4)

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  1. U PBM -Gent Polymer Chemistry & Biomaterials Group UGent contribution to PolExGene(WP 2, 3 & WP 4) PolExGene Technical Meeting Tübingen 18-19/05/2009 PolExGene V.Vermeersch, S. Van Vlierberghe, V. TonchevaP. Dubruel

  2. Workpackage Contents Workpackage 2: Development of CPP-containing polymers Reference polymers Specific polymers: polyglutamine derived homo- & copolymers Coupling to fluorescent markers, cell penetrating peptides Workpackage 3:Development of CIP containing polymer membranes Development of polymer membranes by solvent-casting and electrospinning Characterisation through AFM, SEM, XPS, ATR-FTIR, TOF-SIMS Workpackage 4:Preparation of DNA and CPP-containing polyplexes Examining condensing capacity of the polymers Different aspects of polyplex formation V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 2

  3. Summary (Dec 2008) • Synthesis of poly-L-Glutamine derivatives - Homopolymers (-NMe2) - Co-polymers (-NMe2 & -NH2) - Co-polymers (-NMe2 & -NH2 & -OH) - Co-polymers (-NH2/guanidine & -OH) • Synthesis of poly-L-Arginine derivatives - Homopolymers (guanidine) - Copolymers (guanidine & -NH2) • Synthesis of poly-L-Lysine derivatives - Homopolymers (-NH2) - Modified homopolymers (guanidine) Mw = 79000/242000 Mw = 5500/27000/67000/113000/242000 Different compostion % Different compostion % Mw = 13000/23000/87000/113000/150000 Mw = 64000 Mw = 50000/130000 Mw = 50000/130000 V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 3

  4. Summary (Dec 2008) • Coupling of peptides - Penetratine - Arginine8Glutamine-co-polymers (-NMe2 & -NH2) PEI- BiotinylatedGlutamine-co-polymers (-NMe2 & -NH2) • Coupling of fluorescent marker: Oregon Green 488 - Glutamine-co-polymers (-NMe2 & -NH2)- PEI + Lysine + Arginine polymers • Coupling of peptides + fluorescent marker - Glutamine-co-polymer (-NMe2 & -NH2): Arginine8 + Oregon Green 488 - Glutamine-co-polymer (-NMe2 & -NH2): Penetratine + Oregon Green 488 Glutamine-co-polymers (-NMe2 & -NH2) Arginine-co-polymers (guanidine & -NH2) PEI ≠ Mw Variable spacer-lengths Pen + Arg8 ≠ concentrations V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 4

  5. Summary (Dec 2008) Physico-chemical characterisation part • FTIR, UV & 1H-NMR measurments • Molecular weight determination by GPC & viscosimetry • Agarose gel-electrophoresis • EtBr fluorescence • Dynamic light scattering → complex size / distribution • Zeta-potential V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 5

  6. Synthesis Feedback from previous transfection tests: Presence of guanidine groups advantageous effect on transfection (VT01) Amine side groups inefficient (interaction to tight?) Covalent arginine8 beneficial effect on transfection (V14) Improved line of polymers: Preserve the excellent viability by keeping the biodegradable, biocompatible amide-backbone but with adjusted side-chain end-groups • Investigation of the Mw influence • Introducing Imidazole as functional side groups • Further optimising of peptide influence (peptide concentration, changing crosslinker, fluorescently labelled) • Grafting of poly-L-Arginine onto Glutamine-derivatives V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 6

  7. Synthesis Influence of the Mw on transfection? p(DMAEG85%-AEG15%) p(HEG87%-GuEG13%) Mw = 150000 - 27000 Mw = 5500 – 27000 - 67000 – 113000 - 242000 V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 7

  8. Synthesis Introduction of Imidazole functional groups Beneficial for transfection? p(HEG-ImEG) VT09: 40 – 60 % (Mw = 10.000) PositiveTransfection (Higher CR-ratio) To be sent p(HEG-ImEG) p(HEG-AEG-ImEG) V32: 93 – 7 % (Mw = 113.000) V32: 80 – 12 – 8 % (Mw = 26.000) V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 8

  9. Synthesis Grafting of polyarginine onto Glutamine derivatives • Efficient glutamine backbone (87% -OH, 13% -NH2) • Benefical Arg8 CPP effect Cfr. VT02 Cfr. V14a Comparison p(DMAEG-AEG)Mw = 113.000 35 % Grafting p(HEG-AEG)Mw = 27.00050 % Grafting V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 9

  10. Peptide Coupling Optimising CPP effect New crosslinker (SPDP) Higher concentration of Arg8 S-S bond ► Cleavable once internalised Penetratine + SPDP (V33) = 2 % (coupled to glutamine copolymer) SPDP ► V31b: pHEG-AEG + 5 % Arginine8 ► Investigating CPP effect Fluorescent label V26a/V26b V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 10

  11. Methacrylate synthesis Polymethacrylates Proven transfection efficiency (e.g. V04/P34) More hydrophobic backbone NOT Biodegradable Possibly more toxic? Different synthesis Radical polymerisation ► Influence of Mw (Range 50.000 – 800.000) ► Similar polymer compositions pDMAEMA pDMAEG pDMAEMA ► Similar co-polymer compositions p(DMAEMA-AEMA) p(DMAEG-AEG) V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 11

  12. Physico-chemical testing EtBr Fluorescence: Information on polyplexes formation as a function of polymer-concentration (expressed as CR) V31 = p(HEG-AEG) Fluorescence yield pArginine VT02p(HEG-GuAEG) pEI V31a= V31+Arg-grafts CR V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 12

  13. Future plans • Synthesis of ‘improved’ polymers? - Comparison methacrylate – glutamine (-OH & -NR2) - Requested synthesis? • Coupling of peptides - SPDP coupling with –OH containing polymers - Requested synthesis? • Continued Fluorescence • Continued Dynamic Light Scattering • Continued Zeta-potential • Degradation studies (lysozyme) V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 13

  14. PBM products sent-out V. Vermeersch – PolExGene Tübingen – 18/05/2009 pag. 14

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