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PolExGene – 18 month midterm meeting 18.12.2007

Heiko Büth. PolExGene – 18 month midterm meeting 18.12.2007. Overview. WP4 - Preparation of plasmids and CPP-containing polyplexes. Formation kinetics of polyplexes using Fluorescence Correlations Spectroscopy (FCS):. Determination of diffusional properties.

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PolExGene – 18 month midterm meeting 18.12.2007

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  1. Heiko Büth PolExGene – 18 month midterm meeting18.12.2007

  2. Overview • WP4 - Preparation of plasmids and CPP-containing polyplexes

  3. 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

  4. 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

  5. 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

  6. Forming of polymer and DNA polyplexes: 1. Alternative: Addition of polymer to a DNA solution 2. Alternative: Addition of DNA to a polymer solution 2 1 • Solution consists of: • few highly loaded plasmid-DNA strands with extreme charge ratios • naked plasmid-DNA strands • Solution consists of: • plasmid-DNA with polymers in an equally distributed charge ratio

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

  8. 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 • 

  9. FCS measurements of polyplex formation kinetics: • 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 • 

  10. FRET MM-QDot Dye-Emission FRET Qdot-Emission Excitation • FRET measurements of CPP position within the polyplex: • fluorescence labeled CPP as acceptor • quantum dot as donor

  11. FRET measurements of CPP position within the polyplex: • Determination of position of CPP within the polyplex: • lipid coated quantum dots are not available on the market • quantum dots are currently synthesized • change to streptavidin biotin detection system as alternative

  12. Future plans: • Further FCS and FCCS analysis of different polyplexes • Determination of CPP localization within polyplex

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