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UGent contribution to PolExGene (WP 2 & WP 4) PolExGene Technical Meeting Prague 22-23/05/2008 PBM G ent - G entU Polymer Chemistry & Biomaterials Group.

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Presentation on theme: "UGent contribution to PolExGene (WP 2 & WP 4) PolExGene Technical Meeting Prague 22-23/05/2008 PBM G ent - G entU Polymer Chemistry & Biomaterials Group."— Presentation transcript:

1 UGent contribution to PolExGene (WP 2 & WP 4) PolExGene Technical Meeting Prague 22-23/05/2008 PBM G ent - G entU Polymer Chemistry & Biomaterials Group V.Vermeersch, S. Van Vlierberghe, V. Toncheva P. Dubruel & E. Schacht

2 Overview Workpackage contents Summary of past work Work performed Ongoing & future plans V. Vermeersch – PolExGene Prague – 22/05/2008 pag. 2

3 Workpackage Contents pag. 3 Workpackage 2: Development of CPP-containing polymers Workpackage 4: Preparation of DNA and CPP-containing polyplexes Reference polymers Specific polymers: poly-  -aminoacid derived homo- & copolymers Coupling to fluorescent markers, cell penetrating peptides Examining the physico-chemical properties of polymers (condensing capacity) 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 V. Vermeersch – PolExGene Prague – 22/05/2008

4 Synthesis-part  Synthesis of Poly-L-Glutamine derivatives  Homopolymer (V01-V03) & Copolymer (V05-V06)  Comparison to reference polymers - PEI & PDMAEMA (V04)  Poly-L-Glutamine + fluorescent marker (Oregon Green ® 488) (V07a/b/c-V08)  PEI + fluorescent marker (Oregon Green ® 488)  Poly-L-Glutamine + CPP (Penetratine) (V09a/b-V10)  Synthesis of Arginine-NCA (R-NCA) & poly-L-Arginine  Grafting of Poly-L-Arginine on Poly-L-Lysine pag. 4 Summary Ghent Meeting (Dec 2007) V. Vermeersch – PolExGene Prague – 22/05/2008

5  IR, UV & NMR measurments (V01-V12)  Molecular weight determination by GPC & viscosimetry (M v & M w of V01-V06)  Agarose gel-electrophoresis Determination of CR’s; testing of polyplex stability in different environments (H 2 O; NaCl; Cell medium) (V01-V10)  EtBr fluorescence (V01-V10) Determination CR-profile; examining kinetics of polyplexformation  Dynamic light scattering → complex size / distribution all samples smaller than 200 nm (< 100 CR = 4/1) (V01-V06) Physico-chemical characterisation part pag. 5 Summary Ghent Meeting (Dec 2007) V. Vermeersch – PolExGene Prague – 22/05/2008

6 Continued Synthesis pag. 6 Synthesis based on Arginine-NCA (R-NCA) Low yield Side reactions complex purification Synthesis of Poly-L-Arginine based on Poly-L-Ornithine (Ghent-meeting target) V. Vermeersch – PolExGene Prague – 22/05/2008 Alternative strategy

7 Continued Synthesis pag. 7 V. Vermeersch – PolExGene Prague – 22/05/2008 Conversion into Poly-L-Arginine: specific ‘guanidylating’ agent (97%) Polyornithine 3,5-dimethyl-1- guanylpyrrazole Polyarginine 3,5-dimethylpyrrazole Characterisation - M v (poly-N-CBZ-Ornithine) in CHCl 2 COOH - NMR & IR spectra - M w (Gel Permeation Chromatography) Method of analysis currently optimised (GPC detector down) Range of M/I ratio’s 5/1 – 10/1 – 25/1 – 100/1 – 250/1 M w = ?

8 GMBS Intermediate: GMBS coupling Peptide Coupling pag. 8 CPP-coupled polymer pDMAEG-AEG-CPP (V09/V10) V. Vermeersch – PolExGene Prague – 22/05/2008 NH 2 -polymer Thiol-coupling Thiolated CPP’s (Montpellier) Penetratine (available)  V09 (1,65% & 2,79%)  V10 (1,77%)  PEI (0,47%) Octa-Arginine  No spacer  CH 2 -moiety spacer (3/5/7)  PEG spacer (2/11) RGD CycloRGD

9 Physico-chemical testing  Agarose Gel-electrophoresis: Condensation point V11-V12-V13 (in nm)CR0h48h V13a *466* V12V11 DNA CR 1.0 CR 1,8 V13a  Dynamic Light Scattering (DLS) (in nm)CR0h48h V13b EtBr Fluorescence pag. 9 V. Vermeersch – PolExGene Prague – 22/05/2008

10 Physico-chemical testing Time (s) Fluorescence yield Single-shot kinetics (2-1 CR) pag. 10 V. Vermeersch – PolExGene Prague – 22/05/2008

11 Future plans  Synthesis of combined polymers - Guanidine-Glutamine - Guanidine-PEI - Guanidylated PLL  Coupling of Octa-Arginine - Polyglutamine copolymers (V05, V06) - Polyarginine (V13) - PEI - CPP density variation  Zeta-potential measurments of V09-V13 (+ V13 derivatives)  DLS of CPP-coupled polymers (Polymers with free –NH 2 ) pag. 11 V. Vermeersch – PolExGene Prague – 22/05/2008

12 PBM products sent-out GROUPV13a/bVT VT07 2/3/4VT09/10 ENSx/xx/x/x/xx/x/xx/x UH.FPx/xx/x/-/-x/-/-x/x IBMT-/xx/x/-/- x/- UKU ARK UAT IMIC-/xx/x/-/- x/- CNRS pag. 12 V. Vermeersch – PolExGene Prague – 22/05/2008


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