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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 Final Meeting Ghent 19-20/11/2009. PolExGene. V.Vermeersch , V. Toncheva, P. Dubruel. Workpackage Contents. Workpackage 2: Development of CPP-containing polymers.

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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 Final Meeting Ghent 19-20/11/2009 PolExGene V.Vermeersch, V. Toncheva, P. 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 Ghent – 19/11/2009 pag. 2

  3. Summary • Synthesis of poly-L-Glutamine derivatives - Homopolymers (-NMe2) - Co-polymers (-NMe2 & -NH2) - Co-polymers (-NH2 & Guanidine & -OH) - Co-polymers (-NH2 & -OH & Imidazol) • Synthesis of poly-L-Arginine derivatives - • Synthesis of poly-L-Lysine derivatives - Modified homopolymers (guanidine) • Grafting of poly-L-Arginine on poly-L-Glutamine (via –NH2) - Grafting on (–OH/NH2) and (–NMe2/NH2) polymer Mw = 79000/242000 g/mol Mw = 5500-242000 g/mol Different compostion % Different compostion % Mw – Range : 5000 – 150000 g/mol Mw = 50000/130000 g/mol ≠% Arginine V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 3

  4. Summary • Coupling of peptides - Penetratine - Arginine8≠ Mw polymers; ≠ Amounts coupled; Variable spacer lengths- Biotinylated - Different crosslinkers: GMBS or SPDP • Coupling Fluorescent marker: Oregon Green 488/Alexa Fluor 544 • Coupling of peptides + Fluorescent marker - Arginine8/Penetratine + Oregon Green 488 ≠ Mw polymers; ≠ Amounts coupled Non-Biodegradablepolymers: polymethacrylates Comparison biodegradable poly-L-Glutamine≠ Mw (range: 50.000 – 800.000 g/mol) CH3 (CH2-C) R V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 4

  5. Summary Chemical characterisation • FTIR, UV & 1H-NMR measurements Physico-chemical characterisation • Molecular weight determination by GPC & viscosimetry • Agarose gel-electrophoresis • EtBr fluorescence • Dynamic light scattering → complex size / distribution • Zeta-potential V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 5

  6. Synthesis Effect of CPP on ‘Active-like’ polymer (cfr. VT02) Coupling Penetratine (2,5 %) Coupling Arginine8 (4,5 %) SPDP crosslinker(cleavable intra-cellular) Comparison: Arginine8 Arginine-grafting Sent to be tested out (ENS) V31b/V31c V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 6

  7. Synthesis Effect of guanidine-amount on ‘Active-like’ polymer (cfr. VT02) ? 58 10.000 42 V31g = not active (Mw too high?) V35g/V35g-2 = sent out for testing To be produced: guanidine 40-50% V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 7

  8. Synthesis Effect of cationic group on ‘Active-like’ polymer (cfr/ VT02) Hydroxyl present (≥ 50 %) Dimethylaminoethyl group (NMe2) NMe2/OH V36a: 21 – 79 % (Mw = 10.700) V36b: 40 – 60 % (Mw = 11.100) V36c: 67 – 33 % (Mw = 11.900) p(HEG-DMAEG) Cationic group: NMe2 & Guanidine Polymers sent (ENS) ; to be tested via rapid screening V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 8

  9. Methacrylate synthesis Polymethacrylates More hydrophobic backbone NOT Biodegradable Toxic? Mw Radical polymerisation ► Similar polymer compositions pDMAEMA OK pDMAEMA pDMAEG In progress p(DMAEMA-HEMA) p(DMAEG-HEG) p(GuAEMA-HEMA) p(GuAEG-HEG) To be done V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 9

  10. Physico-chemical testing EtBr Fluorescence: influence of guanidine contents in DNA-condensation pHEG-GuAEG 13 % Guanidine 25% Guanidine Fluorescence yield pEI 33% Guanidine 37% Guanidine CR V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 10

  11. Physico-chemical testing EtBr Fluorescence: influence of guanidine ‘orientation’ in DNA-condensation ≈ amount guanidine Poly-Arginine (homopolymer) pEI Fluorescence yield Poly-Arginine grafting (50%) pHEG-GuAEG Arg8 CPP(4,5%) CR V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 11

  12. Physico-chemical testing DLS: 1/1 2/1 4/1 No complexation Mean: 50,3 nm Mean : 46,7 nm Agarose Gel-Electrophoresis V35g V35g-2 Step 0,2 Step 0,2 DNA DNA 2/1 2/1 V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 12

  13. Future plans • Finalising ongoing synthesis- Comparison methacrylate – glutamine - pHEG-GuAEG compound with  amount of guanidine • Requested synthesis • Finalising EtBr Fluorescence, DLS & Agarose-electrophoresis • Zeta-potential of active and ‘active-like’ polymers • Degradation studies (lysozyme) • Nuclease studies • Hemo-agglutination studies Active polymers Reporting & publications V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 13

  14. PBM products sent-out V. Vermeersch – PolExGene Ghent – 19/11/2009 pag. 14

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