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PolExGene – 18 month midterm meeting 17.12. - 18.12.2007, Ghent, Belgium

Heiko Büth. PolExGene – 18 month midterm meeting 17.12. - 18.12.2007, Ghent, Belgium. 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)

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PolExGene – 18 month midterm meeting 17.12. - 18.12.2007, Ghent, Belgium

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  1. Heiko Büth PolExGene – 18 month midterm meeting17.12. - 18.12.2007, Ghent, Belgium

  2. 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

  3. Selection of CIP and CPP (WP1):Electrical impedance monitoring of membrane integrity on single cells

  4. Selection of CIP and CPP (WP1)Development of cavity chip – Prototype

  5. Selection of CIP and CPP (WP1)Measurement of CPP - penetratin Application of 25 µM penetratin

  6. Selection of CIP and CPP (WP1)Measurement of CPP - penetratin Application of 50 µM penetratin

  7. Selection of CIP and CPP (WP1)Measurement of CPP - penetratin • Test for membrane integrity after CPP incubation - Application of Triton-X 100: • impedance decreases to no-cell level

  8. Selection of CIP and CPP (WP1)Measurement of CPP - penetratin • Impedance measurements on single cells using penetratin: • effects of penetratin are detectable and monitoring over time is possible • impedance decreases 3k after incubation with 10 to 50 µM penetratin • cellular membranes are intact after incubation

  9. Selection of CIP and CPP (WP1)Detection of internalized penetratin • Inspection of cellular uptake of rhodamine-B functionalized CPP: • penetratin accumulates inside positioned single cells

  10. Selection of CIP and CPP (WP1)Measurement of CPP - penetratin • Impedance measurements on single cells using penetratin: • monitoring of penetratin effects on single cells over time • cells are vital • penetratin accumulates inside positioned single cells

  11. 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

  12. Formation kinetics of polyplexes using Fluorescence Correlations Spectroscopy (FCS): Determination of diffusional properties polymer: V8 (FW: 242kDa, Oregon Green labeling degree 5%) plasmid: pCMV (7.2 kbp) CPP: penetratin-Rhodamin-B

  13. FCS measurement of polymer V8 Oregon Green label is suitable for FCS  (Diffusion) = 398 µs

  14. FCS measurement of polymer V8 and DNA (10 nM V8) = 398 µs (5 nM V8 + 5nM DNA) = 1500 µs (30 nM V8 + 5nM DNA) = 2800 µs Formation of polyplex occurs Polyplex formation is concentration dependant

  15. Fluorescence intensity: Variant: low main level, high peaks Variant: constant main level Distribution is stable for more than 24h Forming of polymer and DNA polyplexes: Alternative: Addition of polymer to an DNA solution Alternative: Addition of DNA to an polymer solution • Very fast reaction • no equilibrium • decomposition not detectable by FCS • reaction is controlled by formation kinetics and not by stability

  16. Forming of polymer and DNA polyplexes (Hypothesis): Alternative: Addition of polymer to a DNA solution

  17. Forming of polymer and DNA polyplexes (Hypothesis): Alternative: Addition of polymer to a DNA solution

  18. Forming of polymer and DNA polyplexes (Hypothesis): Alternative: Addition of polymer to a DNA solution

  19. Forming of polymer and DNA polyplexes (Hypothesis): Alternative: Addition of polymer to a DNA solution • Solution consists of: • few highly loaded plasmid-DNA strands with extreme charge ratios • naked plasmid-DNA strands

  20. Forming of polymer and DNA polyplexes (Hypothesis): 2. Alternative: Addition of DNA to a polymer solution

  21. Forming of polymer and DNA polyplexes (Hypothesis): 2. Alternative: Addition of DNA to a polymer solution

  22. Forming of polymer and DNA polyplexes (Hypothesis): 2. Alternative: Addition of DNA to a polymer solution

  23. Forming of polymer and DNA polyplexes (Hypothesis): 2. Alternative: Addition of DNA to a polymer solution • Solution consists of: • plasmid-DNA with polymers in an equally distributed charge ratio

  24. FCS measurements of CPP: • rhodamine-B labeled penetratin is detectable • (penetratin) = 36 µs

  25. FCS measurements of polyplex formation: • molar ratios up to [CPP]/[V8] ~0,4 only moderate binding • (20 nM CPP 200 nM V8) = 100 µs • (40 nM CPP 100 nM V8) = 200 µs • molar ratios up to [CPP]/[V8] ~1.6 • strong formation of polyplexes • (80 nM CPP 50 nM V8) = 4000 µs • 

  26. FCS measurements of polyplex formation: • Formation of polyplexes is detectable by using FCS • Formation of polyplexes is concentration dependent • Order of preparation is important • dilution of plasmid in polymer • influence on transfection efficiency has to be investigated • 

  27. 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

  28. Characterisation of polyplex-cell and polymer membrane-cell interactions (WP5) Developed impedance analysis system will be used to analyze polyplexes in cellular systems • We need: • different CPP • therapeutic plasmids • cells for cellular systems

  29. Month 18 to month 24 • FCS and FCCS analysis of different polyplexes • FRET measurements on CPP localization • Further development of cavity chip • enhanced design based on obtained results • Usage of impedance technology to support evaluation of CPP and CIP using impedance spectroscopy • Usage of impedance technology to support evaluation of polyplexes in cellular systems

  30. Exchanged Products • Received • PEI (UGent) • Sample Polymers (UGent) : • labeled PEI, V08 • Plasmid (UH.FP) • CPP (CNRS) • penetratin, labeled penetratin

  31. Thankyou

  32. Selection of CIP and CPP (WP1):Equivalent circuit model for impedance measurements

  33. 0 µM 250 µM 500 µM 50 µm Selection of CIP and CPP (WP1)Proof-of-concept impedance changes on attachment of single cell L929 (mouse fibroblast) cells in suspension attached by suction force to capillary Diameter of capillary is 8 µm

  34. Selection of CIP and CPP (WP1)Development of cavity chip – Prototype(s)

  35. 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

  36. Selection of CIP and CPP (WP1)Measurement of CPP penetratin Application of 500 µM PEI

  37. Selection of CIP and CPP (WP1)Development of cavity chip – Prototype(s) Application of 1 mM PEI

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