1. UGent contribution to PolExGene (WP 2 & WP 4) PolExGene Technical Meeting Ghent 17-18/12/2007 PBM G ent - G entU Polymer Chemistry & Biomaterials Group V.Vermeersch, S. Van Vlierberghe, P. Dubruel & E. Schacht

2. Overview Workpackage contents Summary of last presentations Work performed Ongoing & future plans V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 2

3. Workpackage Contents V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 3 Workpackage 2: Development of CPP-containing polymers Workpackage 4: Preparation of DNA and CPP-containing polyplexes Reference polymers Specific polymers: polyglutamine derived homo- & copolymers Coupling to fluorescent markers, cell penetrating peptides Examining condensing capacity of the polymers Different aspects of polyplex formation 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

4. Synthesis-part Synthesis of pDMAEG homopolymer (V01 & V03) Synthesis of p(DMAEG-co-AEG) copolymer (V05 & V06) Comparison to reference polymers PEI & PDMAEMA (V04) Coupling to fluorescent marker (Oregon Green ® 488) (V05) Coupling to GMBS crosslinker (V05) Synthesis of Arginine-NCA (R-NCA) Preliminary synthesis of polyarginine V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 4 Summary Helsinki Meeting (Aug 2007)

5.  Molecular weight determination by GPC & viscosimetry  Agarose gel electrophoresis determining condensing CR’s; testing of polyplex stability in different environments.  EtBr fluorescence determining condensing CR’s; examining kinetics of polyplex formation  Dynamic light scattering → measuring of complex size all samples smaller than 200 nm (up to < 100 nm @ CR = 4) Ongoing physico-chemical characterisation of V01-V06 using: V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 5 Summary Helsinki Meeting (Aug 2007)

6. Continued Synthesis V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 6 Homopolymers prepared: pDMAEG M w = 79900 (V01) & 242000 (V03) Copolymers prepared: p(DMAEG 85% -co-AEG 15% ) M w = 91600 (V05) & 242000 (V06) Coupling to fluorescent groups, thiol-selective groups & peptides pDMAEG Tertiary amine: no coupling possible

7. Fluorescent Marker Coupling V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 7 Oregon Green 488 (V07a/b/c) 1 mol% 6,2 mol% 7,3 mol% Fluorescent marker V05 + Fl. = V07 V06 + Fl. = V08 (V08) 5,2 mol% Sent to ENS/UH.FP + (IBMT only V07a) PEI+OREG 2,7 mol%

8. GMBS Covalent linking to Ac-Cys-Penetratine Intermediate: GMBS coupling Peptide Coupling V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 8 CPP-coupled p(DMAEG-co-AEG) V09 = V05 + CPP (2,5 mol%)

9. V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 9 Other Synthesis Other coupling reactions planned: V05 & V06 Varying the CPP density on the polymers H-(Arg) 8 -NH 2 peptide (non-covalently, physically mixed) Newly developed synthesis of polyarginine NCA Arginine polyarginine Initiator: Bu 3 NH 2 Coupling via cysteine-function Limited M w (3000 – 7000 Da) (when using Bu 3 NH 2 ) pLL as initiator pLL-pArg (pLL of various Mw possible) polyarginine grafts ranging from 18000-35000 Da Alternative strategy using polyornithine currently being started

10. Physico-chemical testing Gel electrophoresis: Condensing capacity of all polymers checked Stability of the polyplexes was also confirmed (H 2 0, NaCl & Medium) Ethidium Bromide fluorescence Condensation profile established for all polymers (no V06) (kinetics) V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 10

11. Dynamic Light scattering V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 11 Dynamic light scattering results (DLS) - All polyplexes sizes and distributions were investigated - Size as function of time was checked Low CR’s (close to CCR) distinct aggregation Higher CR’s further condensation (smaller ø) - Size in cell-culture medium was checked (+ in function of time) At high CR’s only moderate aggregation (other CR’s complete agg.) CRpDMAEMA (V04)pEIpDMAEG (V01) 1/1No condensation146,3 ± 12,2427,4 ± 47,2 2/1248,8 ± 4,3107,6 ± 44,3166,4 ± 3,4 4/180,1 ± 3,783,1 ± 0,887,0 ± 3,6 CRpDMAEG (V03)pDMAEG-pAEG (V05)pDMAEG-pAEG (V06) 1/1533,2 ± 28,2No condensation 2/1182,9 ± 4,545,1 ± 3,832,1 ± 5,2 4/161,8 ± 3,956,2 ± 0,931,2 ± 4,7

12. Zeta-potential V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 12 Zeta-potential: measuring charge-difference of a particle in solution with it’s surrounding environment (by measuring it’s stabilizing ion-double-layer)

13. Future plans V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 13 Synthesis of polyarginine (different M w ) - Polyornithine strategy - PLL grafting (various PLL M w try-outs) - Polyagmatine as polyarginine mimic Coupling of other CPP to copolymers (V05, V06) and varying the CPP density on the polymers Comparison of Octa-arginine & penetratine physical mixing Testing of polyplex stability by gel electrophoresis conform optimized ENS/UH.FP transfection protocol Synthesis of Au binding biopolymers to perform SPR measurements (polyplex-ECM interactions)

14. PBM products sent-out GROUPPEIPEI+OREGPDMAEMA (V04)V01V03V05V06V07a/b/cV08V09 ENSxxxxxxxx/x/-xx UH.FPxxxxxxxx/x/-xx IBMT x xx x/-/-x UKU xx ARK UAT IMICx xxxx CNRS V. Vermeersch – PolExGene Ghent - 17/12/2007 pag. 14