1 Status Report about Tasks of IBMT PolExGene – 12 month technical meeting , Helsinki, Finland Heiko Büth

2 PolExGene – 12 month technical meeting Workpackages and Deliverables - IBMT Del. no.Deliverable name WP no. Lead participant Del. date 3Polyplexes and CPP-containing polyplexes41, Transfected retinal/vascular cells using polyplexes 52, 3, 5, Transfected retinal/vascular cells seeded on polymer membranes 52, 3, 5, Cell penetrating peptides (CPP) for polyplex formulations 13, Cell interactive peptides (CIP) for polymer membranes 11, 3, Polyplex containing polymer membranes seeded with retinal/vascular cells using marker genes 52, 3, 5, Polyplex containing polymer membranes seeded with retinal/vascular cells using therapeutic genes 52, 3, 5, 930

3 PolExGene – 12 month technical meeting Preparation of plasmids and CPP- containing polyplexes (WP4) Determination of position of CPP within the polyplex: fluorescence labelled CPP as acceptor quantum dot as donor D3: Polyplexes and CPP-containing polyplexes Determination of polyplex formation kinetics using FCCS: polymer- and DNA-label should not interact labels should not change kinetics of formation (i.e. charge, molecular weight)

4 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Cell penetrating peptides (CPP) for polyplex formulations Cell interactive peptides (CIP) for polymer membranes Electrical impedance monitoring of membrane integrity on single cells:

5 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Proof-of-concept impedance changes on attachment of single cell 50 µm L929 (mouse fibroblast) cells in suspension attached by suction force to capillary Diameter of capillary is 8 µm 0 µM 250 µM500 µM

6 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – first draft Overview simultaneous measurement of four concentrations, reagents etc. quadruplicate measurements easy to fabricate CPP selection 100 µm diameter of gold electrodes 5 µm microholes for application of vacuum CIP selection 100 µm diameter of gold electrodes 5 µm microholes for application of vacuum gold coated surface for binding of CIP

7 PolExGene – 12 month technical meeting Month 6 to month 12  FRET and FCCS analysis of polyplexes  Fabrication of chip with microcavities for single cell analysis fluidic system for positioning of single cells recording electrodes temperature control system  Generation of reference data for evaluation of CPP and CIP using impedance spectroscopy

8 PolExGene – 12 month technical meeting Overview WP1 - Selection of CIP and CPP D10 Cell penetrating peptides (CPP) for polyplex formulations (running) D11 Cell interactive peptides (CIP) for polymer membranes (running) WP4 - Preparation of plasmids and CPP-containing polyplexes D3 Polyplexes and CPP-containing polyplexes (running) WP5 - Characterisation of polyplex-cell and polymer membrane-cell interactions D4 Transfected retinal/vascular cells using polyplexes D8 Transfected retinal/vascular cells seeded on polymer membranes D12 Polyplex containing polymer membranes seeded with retinal/vascular cells using marker genes D15 Polyplex containing polymer membranes seeded with retinal/vascular cells using therapeutic genes

9 PolExGene – 12 month technical meeting Preparation of plasmids and CPP- containing polyplexes (WP4) Determination of position of CPP within the polyplex: lipid coated quantum dots are currently synthesized rhodamine labelled CPP will act as acceptor D3: Polyplexes and CPP-containing polyplexes

10 PolExGene – 12 month technical meeting Preparation of plasmids and CPP- containing polyplexes (WP4) Polyplex FRET Excitation MM-QDot Qdot-Emission Dye-Emission

11 PolExGene – 12 month technical meeting Preparation of plasmids and CPP- containing polyplexes (WP4)  /nm Absorption / Emission EX FRET

12 PolExGene – 12 month technical meeting Overview WP1 - Selection of CIP and CPP D10 Cell penetrating peptides (CPP) for polyplex formulations (running) D11 Cell interactive peptides (CIP) for polymer membranes (running) WP4 - Preparation of plasmids and CPP-containing polyplexes D3 Polyplexes and CPP-containing polyplexes (running) WP5 - Characterisation of polyplex-cell and polymer membrane-cell interactions D4 Transfected retinal/vascular cells using polyplexes D8 Transfected retinal/vascular cells seeded on polymer membranes D12 Polyplex containing polymer membranes seeded with retinal/vascular cells using marker genes D15 Polyplex containing polymer membranes seeded with retinal/vascular cells using therapeutic genes

13 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s)

14 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s)

15 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s) Application of 50 µM PEI

16 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s) Application of 50 µM PEI

17 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s) Application of 250 µM PEI

18 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s) Application of 250 µM PEI

19 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s) Application of 500 µM PEI

20 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s) Application of 500 µM PEI

21 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Development of cavity chip – Prototype(s) Application of 1 mM PEI

22 PolExGene – 12 month technical meeting Selection of CIP and CPP (WP1) Prototype(s) Impedance measurements of single cells and measurement of PEI effect on single cells are possible cells are vital in the test system for more than 10 hours

23 PolExGene – 12 month technical meeting Overview WP1 - Selection of CIP and CPP D10 Cell penetrating peptides (CPP) for polyplex formulations (running) D11 Cell interactive peptides (CIP) for polymer membranes (running) WP4 - Preparation of plasmids and CPP-containing polyplexes D3 Polyplexes and CPP-containing polyplexes WP5 - Characterisation of polyplex-cell and polymer membrane-cell interactions D4 Transfected retinal/vascular cells using polyplexes D8 Transfected retinal/vascular cells seeded on polymer membranes D12 Polyplex containing polymer membranes seeded with retinal/vascular cells using marker genes D15 Polyplex containing polymer membranes seeded with retinal/vascular cells using therapeutic genes

24 PolExGene – 12 month technical meeting Characterisation of polyplex-cell and polymer membrane-cell interactions (WP5) Experiments in WP5 depend on impedance test system and polyplexes developed in WP1

25 PolExGene – 12 month technical meeting Month 12 to month 18  FRET and FCCS analysis of polyplexes  Final development of cavity chip enhanced design based on obtained results optimized fluidic system to move from reflected light to transmitted light microscope independent temperature control system  Usage of cavity chip for evaluation of CPP and CIP using impedance spectroscopy

26 PolExGene – 12 month technical meeting Exchanged Products Received PEI (UGent) Sample Polymers (UGent) : V01, V03, V07 Sent Cavity chips for lipid layer application (UH.FP) Gold coated glass slides for CIP binding studies (UGent)