1 Status Report about Tasks of IBMT PolExGene – 12 month technical meeting 23.08. - 24.08.2007, Helsinki, Finland Heiko Büth.

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Status Report about Tasks of IBMT

PolExGene – 12 month technical meeting

23.08. - 24.08.2007, Helsinki, Finland

Heiko Büth

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Workpackages and Deliverables - IBMT

Del. no. Deliverable name WP no.

Lead participant

Del.date

3 Polyplexes and CPP-containing polyplexes 4 1, 3 12

4 Transfected retinal/vascular cells using polyplexes 5 2, 3, 5, 9 12

8 Transfected retinal/vascular cells seeded on polymer membranes 5 2, 3, 5, 9 18

10 Cell penetrating peptides (CPP) for polyplex formulations 1 3, 5 18

11 Cell interactive peptides (CIP) for polymer membranes 1 1, 3, 5 18

12Polyplex containing polymer membranes

seeded with retinal/vascular cells using marker genes

5 2, 3, 5, 9 24

15Polyplex containing polymer membranes

seeded with retinal/vascular cells using therapeutic genes

5 2, 3, 5, 9 30

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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)

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Selection of CIP and CPP (WP1)

Cell penetrating peptides (CPP) for polyplex formulationsCell interactive peptides (CIP) for polymer membranes

Electrical impedance monitoring of membrane integrity on single cells:

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Selection of CIP and CPP (WP1)Proof-of-conceptimpedance changes on attachment of single cell

50 µm

L929 (mouse fibroblast) cells in suspensionattached by suction force to capillaryDiameter of capillary is 8 µm

0 50 100 150 200 250 300 350

0,85

0,90

0,95

1,00

1,05 Applied PEI 0 250 M 500 M

Nor

mal

ized

Im

peda

nce

mag

nitu

de a

t 1 k

Hz

Time (s)

PEI

0 µM250 µM 500 µM

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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

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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

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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

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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

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Polyplex

FRET

FRET

Excitation

MM-QDot

Qdot-Emission

Dye-Emission

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/nmAbsorption / Emission

EX

FRET

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Overview


• WP4 - Preparation of plasmids and CPP-containing polyplexes• D3 Polyplexes and CPP-containing polyplexes (running)




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

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15



Application of 50 µM PEI

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17




18




19




20




21



Application of 1 mM PEI

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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

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Overview


• WP4 - Preparation of plasmids and CPP-containing polyplexes• D3 Polyplexes and CPP-containing polyplexes




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Characterisation of polyplex-cell and polymer membrane-cell interactions (WP5)

Experiments in WP5 depend on impedance test system and polyplexes developed in WP1

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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

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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)

1 Status Report about Tasks of IBMT PolExGene – 12 month technical meeting 23.08. - 24.08.2007, Helsinki, Finland Heiko Büth.

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