1 status report about tasks of ibmt polexgene – 12 month technical meeting 23.08. - 24.08.2007,...
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1
Status Report about Tasks of IBMT
PolExGene – 12 month technical meeting
23.08. - 24.08.2007, Helsinki, Finland
Heiko Büth
2
PolExGene – 12 month technical meeting
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
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 formulationsCell 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-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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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
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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
FRET
Excitation
MM-QDot
Qdot-Emission
Dye-Emission
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PolExGene – 12 month technical meeting
Preparation of plasmids and CPP-containing polyplexes (WP4)
/nmAbsorption / 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
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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
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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
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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
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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)
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