1 / 12

UGent contribution to PolExGene (WP 2 & WP 4)

U. PBM. - G ent. 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. Overview. Workpackage contents Summary of last presentation Work Performed

ganesa
Télécharger la présentation

UGent contribution to PolExGene (WP 2 & WP 4)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related