UGent contribution to PolExGene (WP 2 & WP 4)

1. U PBM -Gent Polymer Chemistry & Biomaterials Group UGent contribution to PolExGene(WP 2 & WP 4) Polexgene Technical meeting Helsinki 23-24/08/2007 PolExGene V.Vermeersch, P. Dubruel & E. Schacht

2. Overview • Workpackage contents • Summary of last presentation • Work Performed • Ongoing & future plans V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 2

3. Workpackage Contents Workpackage 2: Development of CPP-containing polymers Reference polymers Specific polymers: polyglutamine derived homo- & copolymers Coupling to fluorescent markers, cell penetrating & -interacting 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 Helsinki - 23/08/2007 pag. 3

4. Summary Paris Meeting (Feb 2007) • Synthesis of pDMAEG homopolymer (V01 & V03) • Synthesis of p(DMAEG-co-AEG) copolymer (V05) • Comparison to reference polymers PEI & PDMAEMA (V04) • Ongoing physico-chemical characterization of V01, V03, V04 using: • Molecular weight determination • Gelagarose electrophoresis • EtBr fluorescence • Dynamic light scattering → determining complex size (< 200 nm) Sufficient DNA condensing capacity V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 4

5. Continued Synthesis Homopolymers prepared: pDMAEGMw = 79900 (V01) & 262000 (V03) Copolymers prepared: p(DMAEG85%-co-AEG15%)Mw = 91000 (V05) & 219000 (V06) • V01/V03 sent to: • Paris (ENS) • Helsinki (UH.FP) • Prague (IMIC) • St. Inkbert (IBMT) • Kuopio (UKU) Ongoing analysis • V05 sent to: • Paris (ENS) • Helsinki (UH.FP) Coupling to fluorescent groups, thiol-selective groups & peptides V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 5

6. Coupling synthesis Ongoing coupling: GMBS V05 Oregon Green 488 GMBS 1 mol% Fluorescentmarker (V07) Sent to: IBMT & IMIC Covalent linking to Cys-peptides V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 6

7. Other Synthesis • Coupling reactions planned: V05 & V06 • RGD • CycloRGD • H-(Arg)8-NH2 peptide(non-covalently, physically mixed) Coupling to polymers via cysteine-function NCAArginine • Newly developed synthesis of polyarginine • NCA of Arginine was synthesised • Polymerisation of NCA to polyarginine performed • Several initiating systems will be tested • Copolymer derivatives of polyarginine possible Initiator: Primary amine Improved transfection efficiency assumed polyarginine V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 7

8. pEI pDMAEMA pDMAEG pDMAEG 4/1 pEI pEI pDMAEMA pDMAEMA pDMAEG pDMAEG pDMAEG pDMAEG 4/1 4/1 44.7k 262k 44.7k 262k 44.7k 242k 2/1 2/1 2/1 1/1 1/1 1/1 DNA DNA DNA 20 20 µ µ g/ml g/ml Physico-chemical testing Gel electrophoresis: determining condensing capacity of the polymers Table: Charge ratio’s for complete DNA condensation • Stability of the Polymer-DNA complex was investigated: • H2O • NaCl • Medium No DNA release was observed at several time-points pEI, V01, V03, V04,V05, V06 Complex stability after 4 hours incubation in cell culture medium (37°) V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 8

9. Physico-chemical testing EtBr Fluorescence: another ‘point of view’ for DNA condensation 120 pDMAEMA pEI 110 pDMAEG 44.7k 100 pDMAEG 242k pDMAEG85%-pAEG15% 90 80 70 EtBr fluorescence (%) 60 50 40 30 20 10 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 Charge ratio (+/-) All polymers showed analog results compared to gel electrophoresis V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 9

10. Physico-chemical testing Stability of complexes in the presence of Penetratine At least stable up to 1 hour after mixing with complexes (physical mixing) Other time points are currently under analysis Effect of Penetratine coupling to copolymers on the physico-chemical properties of the polyplexes will soon be studied Dynamic light scattering: determining the size (nm) of the polyplexes at different charge ratios. V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 10

11. Future plans • Synthesis of polyarginine with different Mw (all transfection partners) • Coupling of RGD & cycloRGD to copolymers (V05, V06) • Study of Penetratine incorporation: - physical mixing - covalent coupling to GMBS • Effect of H-(Arg)8-NH2 peptide addition to complex solutions • Synthesis of Oregon green 488 coupling to pEI • Synthesis of biopolymer functionalised polyimide membranes Gelatine, hyaluronic acid,… (ECM derivatives) UAT • Coating of gold discs with cysteine modified Penetratine XPS measurements, contact-angle measurements,… IBMT (functionalisation of gold electrodes) • Development of new membranes for immunological testing IMIC V. Vermeersch – PolExGene Helsinki - 23/08/2007 pag. 11

12. PBM products sent-out • Polymers • PEI, PDMAEMA (V04), PDMAEG (V01, V03), P(DMAEG-co-AEG) (V05) • P(DMAEMA-co-AEMA) (P34) ENS • Fluorescently labelled P(DMAEG-co-AEG) + Oregon Green (V07) • Polymer membranes • Gelatine (10 wt% type B) coated 96-Well plates IMIC • Gelatine (10 wt% type B) coated Transwell plates UH.FP