24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group

24 months Prague meeting

Sixth Framework Programme Priority 1

Life Sciences, Genomics and Biotechnology for Health

Lorenzo TibaldiAlain Joliot’s groupENS Paris

Work package 1: Selection of CIP and CPP

Work package 2: Development of CPP-containing polymers

Work package 4: Preparation of plasmids and CPP-containing polyplexes

Work package 5: Characteriation of polyplex-cell and polymer membrane-cell

interactions

ENS partner workpackages contribution:

Last 6 months tasks:

• Test the transfection efficiencies of new polymers synthetized by UGent (VT0 and V0 series)

• Study the intracellular behaviours of fluorescent polymers and fluorescently labelled plasmid by confocal microscopy on live transfected cells

Test new polymers

CV1 cells5µL complex24hrs incubationRucHA plasmid

VT01 and VT02-2 areactive and efficient

Ruc activity 5µL

-5,0E+04

0,0E+00

5,0E+04

1,0E+05

1,5E+05

2,0E+05

2,5E+05

VT01 VT02-2 VT03 VT04 VT05 VT07-1 pLL V11c V12 V13a V13b PEI

RLU

n.p. 8/1

n.p. 4/1

n.p. 2/1

n.p. 1/1

Viability 5µL

-20%

0%

20%

40%

60%

80%

100%

120%

140%

VT01 VT02-2 VT03 VT04 VT05 VT07-1 pLL V11c V12 V13a V13b PEI

cell viability

n.p. 8/1

n.p. 4/1

n.p. 2/1

n.p. 1/1

Test new polymers

ARPE19 cells10µL complex24hrs incubationRucHA plasmid

VT02-2 is confirmed to beactive also on ARPE

VT09 is active too

Ruc activity 10µL

-5,0E+04

0,0E+00

5,0E+04

1,0E+05

1,5E+05

2,0E+05

2,5E+05

3,0E+05

3,5E+05

4,0E+05

4,5E+05

5,0E+05

VT02-2 VT07-2 VT07-3 VT07-4 VT09 VT10 PEI

RLU

n.p. 8/1

n.p. 4/1

n.p. 2/1

n.p. 1/1

Viability 10µL

-20%

0%

20%

40%

60%

80%

100%

120%

140%

160%


Viability

n.p. 8/1

n.p. 4/1

n.p. 2/1

n.p. 1/1

Test new polymers

ARPE19 cells5µL complex24hrs incubationRucHA plasmid

VT02-2 is confirmed to beactive also on ARPE

VT09 is active too

Ruc activity 5µL

-1,0E+05

0,0E+00

1,0E+05

2,0E+05

3,0E+05

4,0E+05

5,0E+05

6,0E+05


RLU

n.p. 8/1

n.p. 4/1

n.p. 2/1

n.p. 1/1

Viability 5µL

-20%

0%

20%

40%

60%

80%

100%

120%

140%


Viability

n.p. 8/1

n.p. 4/1

n.p. 2/1

n.p. 1/1

Conclusions on new polymers:

•All the members of the VO serie of polymers tested in the last 12 months are inactive

•VT01 and VT09 are (biodegradable?) guanylated variants of PEI and are active

• VT02-2 represents a new biodegradable active polymer

Aims of this technique:

• Compare the cellular behaviour of Oregon Green labelled PEI (active) and V0 polyplexes (inactive)

• Follow the intracellular localization of fluorescent polymers and fluorescent plasmids

• Correlate intracellular polyplex behaviours with reporter protein expression

Confocal microscopy studies of fluorescent polyplexes intracellular behaviours

Transfection activity of fluorescent polyplexes

sRuc SN supernatant 10µL

0,0E+00

5,0E+04

1,0E+05

1,5E+05

2,0E+05

2,5E+05

3,0E+05

3,5E+05

4,0E+05

np 3/1 np 2,5/1 np 2/1 np 1,5/1 np 1/1

PEI

PEI-fluo UGent

PEI-Oregon Green

QuickTime™ et undécompresseur TIFF (LZW)

sont requis pour visionner cette image.



0,25 0,375 0,5 0,75 1 1,5 2 3 0,25 0,375 0,5 0,75 1 1,5 2 3

PEI-Oregon Green PEI-fluo UGentPEI0,25/1 0,5/1 1/1 DNA

Transfection activity of fluorescent polyplexes

sRuc Supernatant 10µL

0,0E+00

2,0E+05

4,0E+05

6,0E+05

8,0E+05

1,0E+06

1,2E+06

np 4/1 np 3/1 np 2,5/1 np 2/1 np 1,5/1

PEIPEI-DNAsytoxG 5µMPEI-Oregon

DNA 0,25 0,5 1/1 0,25 0,32 0,5 0,75 1/1 1,5 2

PEI PEI-Oregon

DNA 0,25 0,5 1/1 0,25 0,32 0,5 0,75 1/1 1,5 2

PEI PEI-Oregon

control plasmid sytox labelled plasmid

Compare internalization efficiency and intracellular behavioursof Oregon Green labelled PEI and V0 polymers (inactive)

Ruc activity 24hrs after transfection

0,0E+00

2,0E+05

4,0E+05

6,0E+05

8,0E+05

1,0E+06

1,2E+06

1,4E+06

1,6E+06

1,8E+06

PEI LF2000 V07

RLU





PEI-Oregon Green without DNA

imaged at 3h

add Blue Trypan

Cell surface fluorescentstaining is quenched byBlue Trypan

Distinguish between extraand intracellular PEI

PEI-Oregon Green complexed with labelled DNA

1h 4hrs 24hrs

+BT

PEIDNAWGA

V07 complexed complexed with labelled DNA

1h 4hrs 24hrs

+BT

PEIDNAWGA

PEI and V07 complexes have different sizes

PEI V07

Follow the intracellular localization of fluorescent polymers and fluorescent plasmids separately

PEI-Oregon complexed with labelled DNA

1h

4hrs

24hrs

+BT

merge PEI plasmid

Dead cells

Correlate intracellular polymer/plasmid behaviours with reporter protein expression

Early protein expression (4hrs)after transfection could help usin correlating polymer/DNA intracellular localization withtransfection activity!

Ruc Expression (dose 10µL)

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5 6 7

hours polyplex incubation

RLU

PEI-Oregon 2,5/1

To do in the next 6 months:

Using the optimized conditions tested by UHFP we want to:

• Test the correlation between transfection activity of the polyplexes and their intracellular behaviours at different time points (expression of GFP or HaloTagged proteins)

• Use high PEI n.p. ratios to understand if toxicity affects all the treated cells or if it is induced particularly in the transfected ones

• Test the intracellular distributions of new biodegradable polymers synthetized by Ugent

• Test the effects on polyplex behaviours and on kinetics of internalization and intracellular distribution of CPP-linked polymers

Documents

24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group