UGent contribution to PolExGene (WP 3 & WP 4) Polexgene Technical meeting Prague 22-23/05/2008 PBM G ent - G entU Polymer Chemistry & Biomaterials Group S. Van Vlierberghe, V. Vermeersch, P. Dubruel & E. Schacht

Overview Workpackage contents Summary of last presentation Planning since previous meeting Work Performed Ongoing & future plans S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 2

Workpackage Content S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 3 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 plasmids and CPP-containing polyplexes Study of the coating of polymer membranes with CPP-containing polyplexes

Summary Ghent meeting (Dec 2007) S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 4 PI grafted with gelatin type B 1.Aminopropylmethacrylamide + gel-MOD 2.Succinimidylester of AFB + gelatin Characterization of grafted PI 1.ATR-IR spectroscopy 2.Atomic force microscopy (AFM) 3.X-ray photo-electron spectroscopy (XPS) 4.Static contact angle measurements (SCA)

Preparation of polymer membranes based on gelatin type A (IEP = 8) (IEP gelatin B = 5). Preparation of polymer membranes based on gel-MOD + HA-MOD (1 – 5 w/v%) gel-MOD + CS-MOD (1 – 5 w/v%) SPR measurements: Study of interaction between gel-MOD (A, B) and polyplexes. WP 4: Coating of PI membranes with CPP-containing polyplexes. Coating of gold chips with CPP’s and CIP’s → impedance Modification of gelatin with DTPA (to chelate technetium) Coating of bare stents with gelatin-DTPA and gelatin + glycerol Planning since previous meeting S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 5

Preparation of polymer membranes based on gelatin type A (IEP = 8) (IEP gelatin B = 5). Preparation of polymer membranes based on gel-MOD + HA-MOD (1 – 5 w/v%) gel-MOD + CS-MOD (1 – 5 w/v%) SPR measurements: Study of interaction between gel-MOD (A, B) and polyplexes. WP 4: Coating of PI membranes with CPP-containing polyplexes. Coating of gold chips with CPP’s and CIP’s → impedance Modification of gelatin with DTPA (to chelate technetium) Coating of bare stents with gelatin-DTPA and gelatin + glycerol Planning since previous meeting S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 6

PI grafted with gelatin type A S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 7 PI aminopropylmethacrylamide gel-MOD (type A) MeOH, 21°C 5 w/v% gel (type A)

Characterization of PI + gel-MOD A S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 8 ATR-IR spectroscopy Gelatin A characteristic peaks: 1629 cm -1, 1538 cm -1 IR-peaks (PI + gel-MOD type A): 1622 cm -1, 1547 cm -1

Porous PI S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 9 PI PI + gel-MOD 1 µm

Coating of gold chips with CIP’s/CPP’s S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 10 Cyclo-RGD: 1634 cm -1, 1544 cm -1 Au + cyclo-RGD: 1663 cm -1, 1545 cm -1 RGD: 1640 cm -1, 1528 cm -1 Au + RGD: 1680 cm -1, 1541 cm -1 Pen-SH: 1648 cm -1, 1531 cm -1 Au + pen-SH: 1652 cm -1, 1558 cm -1 → chemisorption (10 mM)

Gelatin modification with DTPA S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 11 Chelation of Gd or Tc => visualization of gelatin coating on stents gelatin diethylenetriaminepenta- acetic acid dianhydride gelatin-DTPA pH 7, 37°C

Characterization using IR-spectroscopy S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 12 IR-spectrum of gelatin-DTPA: 1091 cm -1 and 1630 cm -1 => coupling of DTPA to gelatin

1 H-NMR spectroscopy S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 13 N-CH 2 -COO - Val, Leu, Ile 5% → 30% DS (gel-MOD) = 65%

Gelatin affinity for polyplexes (SPR) S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 14 Gelatin B

Gelatin affinity for polyplexes (SPR) S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 15 Gelatin A (+) Gelatin B (-) lower affinity for polyplexes (+) higher affinity for polyplexes (+)

Gelatin immobilization on porous PI => open pores? Reduce sample size of PI to 0.5 mm for implantation in mice Preparation of polymer membranes based on gel-MOD + HA-MOD (1 – 5 w/v%) gel-MOD + CS-MOD (1 – 5 w/v%) WP 4: Coating of PI membranes with CPP-containing polyplexes => Qualitative + quantitative analysis Coating of bare stents with gelatin-DTPA and gelatin + glycerol Modification of biodegradable membranes with gelatin (Vivosorb®) → feasibility study Ongoing and future plans S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 16

PBM products sent-out S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 17 GROUP PI + gel-MOD A/B Irgacure glass + gel-MOD B wp + gel-MOD A/B PI + // PI + AFB ENS UH.FPX IBMT UKU ARK UAT X / - IMIC- / XX / X / XXX / X CNRS