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PolExGene ( Month 24-30). Thomas Wirth University of Kuopio (UKU) Participant #: 9. Work packages. WP4: 4.1. Development of plasmids for polyplex incorporation. 4.4. Functionalisation of polymer membrane with polyplexes WP5: 5.1. Interaction between CPP-containing polyplexes and cells

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polexgene month 24 30

PolExGene(Month24-30)

Thomas Wirth

University of Kuopio (UKU)

Participant #: 9

work packages
Work packages
  • WP4:
    • 4.1. Development of plasmids for polyplex incorporation.
    • 4.4. Functionalisation of polymer membrane with polyplexes
  • WP5:
    • 5.1. Interaction between CPP-containing polyplexes and cells
    • 5.2. Interaction between CIP-containing polymer membranes and cells.
  • WP7:
    • 7.2. Implantation of polymer membranes for cardiovascular diseases.
where are we now
Where are we now ?
  • WP4:
    • 4.1. Development of plasmids for polyplex incorporation.
      • VEGF
      • VEGF mutants
      • IK-17
      • SEAP
    • 4.4. Functionalisation of polymer membrane with polyplexes
      • Production of recombinant proteins
  • WP5:
    • 5.1. Interaction between CPP-containing polyplexes and cells
      • Tested transfection efficiencies of different polyplexes
      • Tested different protocols
      • compared transfection efficiencies of polyplexes to viral vectors
    • 5.2. Interaction between CIP-containing polymer membranes and cells.
  • WP7:
    • 7.2. Implantation of polymer membranes for cardiovascular diseases.
what was planned for months 24 30
What was planned for months 24-30
  • Evaluation of new polyplexes received from UGent
  • Testing of transfection protocol (UH.FP)
  • Sending stents to UGent
  • Evaluation of cell growth on polymers
evaluation of vt01 vt02 2 and vt09 polyplex transfection
Evaluation of VT01, VT02-2 and VT09 polyplex-transfection

Polyplex formation protocol

Transfectionprotocol

  • Different charge ratio’s were prepared with DNA (EPI-SEAP plasmid) and VT01, VT02-2 and VT09 polyplexes
  • Charge ratios 4/1, 2/1 and 1/1 were tested
  • Diluted with Cationic Polymers Stock Solution (2,95 mM)
  • Polyplexes were prepared 2h before transfection
  • Total 600ng DNA was used in each well
  • 15000 cells were planted to 96-well plate
  • The following day cells were washed with 150µl PBS
  • 100µl serum-free medium was added to each well
  • 50µl polyplex-DNA solution was added to the cells
  • Incubate for 1h and 2h
  • Wash with 150µl PBS
  • Add 100µl full growth medium (F12/DMEM + serum + penstrep)
  • Incubate for 72h before measurement
measurement chemiluminescent seap assay great escape seap clonetech
Measurement: Chemiluminescent SEAP Assay (Great EscAPe SEAP, Clonetech)
  • Start with 50µl cell culture medium (72h incubation after transfection)
  • briefly centrifuge and take supernatant to measurement (15µl)
  • Add 45µl 1x Dilution Buffer
  • Incubate 30min at +65ºC -> cool samples by placing on ice 2-3min
  • Add 60µl Assay Buffer and incubate 5min, RT
  • Add 60µl fresh CSPD Substrate working dilution and incubate for 10min
  • Measure with VectorII Luminometer
vt01 polyplex
VT01 polyplex
  • 2 Different time points and 3 different charge ratios were tested.
  • Charge ratio 4/1 didn’t give any effect on transfection
  • Charge ratio 2/1 worked with 2h transfection but not with 1h
  • Charge ratio 1/1 gave best transfection.
  • 1h transfection gave better transfection efficacy than 2h
vt02 2
VT02-2
  • Only 4/1 charge ratio showed any transfection efficiency.
  • 2h transfection time was superior compared to 1h.
slide9
VT09
  • Transfection efficiency was poor using VT09.
  • Only low levels of SEAP was measured from cell medium under any condition used.
conclusions
Conclusions
  • VT01
    • Transfection was efficient with 1h transfection and 1/1 charge ratio
  • VT02-2
    • Transfection was efficient with 2h transfection and 4/1 charge ratio
  • VT09
    • Poor transfection using VT09
what we will do
What we will do
  • WP4:
    • 4.1. Development of plasmids for polyplex incorporation.
      • VEGF
      • VEGF mutants
      • IK-17
      • SEAP
    • 4.4. Functionalisation of polymer membrane with polyplexes
      • Production of recombinant proteins
  • WP5:
    • 5.1. Interaction between CPP-containing polyplexes and cells
      • Tested transfection efficiencies of different polyplexes
      • Tested different protocols
      • compared transfection efficiencies of polyplexes to viral vectors
    • 5.2. Interaction between CIP-containing polymer membranes and cells.
  • WP7:
    • 7.2. Implantation of polymer membranes for cardiovascular diseases.