ugent contribution to polexgene (wp 2 & wp 4) polexgene technical meeting prague 22-23/05/2008...
TRANSCRIPT
UGent contribution to PolExGene(WP 2 & WP 4)
PolExGene Technical MeetingPrague 22-23/05/2008
PBM-GGentent
UU
Polymer Chemistry & Biomaterials GroupPolymer Chemistry & Biomaterials Group
V.Vermeersch, S. Van Vlierberghe, V. TonchevaP. Dubruel & E. Schacht
Overview
• Workpackage contents
• Summary of past work
• Work performed
• Ongoing & future plans
V. Vermeersch – PolExGene Prague – 22/05/2008 pag. 2
Workpackage Contents
pag. 3
Workpackage 2: Development of CPP-containing polymers
Workpackage 4:Preparation of DNA and CPP-containing polyplexes
Reference polymers
Specific polymers: poly--aminoacid derived homo- & copolymers
Coupling to fluorescent markers, cell penetrating peptides
Examining the physico-chemical properties of polymers (condensing capacity)
Different aspects of polyplex formation
Workpackage 3:Development of CIP containing polymer membranes
Development of polymer membranes by solvent-casting and electrospinning
Characterisation through AFM, SEM, XPS, ATR-FTIR, TOF-SIMS
V. Vermeersch – PolExGene Prague – 22/05/2008
Synthesis-partSynthesis-part
Synthesis of Poly-L-Glutamine derivatives
Homopolymer (V01-V03) & Copolymer (V05-V06)
Comparison to reference polymers - PEI & PDMAEMA (V04)
Poly-L-Glutamine + fluorescent marker (Oregon Green® 488) (V07a/b/c-V08)
PEI + fluorescent marker (Oregon Green® 488)
Poly-L-Glutamine + CPP (Penetratine) (V09a/b-V10)
Synthesis of Arginine-NCA (R-NCA) & poly-L-Arginine
Grafting of Poly-L-Arginine on Poly-L-Lysine pag. 4
Summary Ghent Meeting (Dec 2007)
V. Vermeersch – PolExGene Prague – 22/05/2008
IR, UV & NMR measurments (V01-V12)
Molecular weight determination by GPC & viscosimetry (Mv & Mw of V01-V06)
Agarose gel-electrophoresis Determination of CR’s; testing of polyplex stability in different environments (H2O; NaCl; Cell medium) (V01-V10)
EtBr fluorescence (V01-V10)
Determination CR-profile; examining kinetics of polyplexformation
Dynamic light scattering → complex size / distribution all samples smaller than 200 nm (< 100 nm @ CR = 4/1) (V01-V06)
Physico-chemical characterisation partPhysico-chemical characterisation part
pag. 5
Summary Ghent Meeting (Dec 2007)
V. Vermeersch – PolExGene Prague – 22/05/2008
Continued Synthesis
pag. 6
Synthesis based on Arginine-NCA (R-NCA) Low yield Side reactions complex purification
Synthesis of Poly-L-Arginine based on Poly-L-Ornithine (Ghent-meeting target)
V. Vermeersch – PolExGene Prague – 22/05/2008
Alternative strategy
* NH CH C
(CH2)3
O
*
NH
n
C
O
O
CH2
Poly-N-CBZ-L-Ornithine
(CH2)2
C O
O
CH2
N-Carbobenzyloxy-L-Ornithine-NCA
O
HN
O
O
* NH CH C
(CH2)3
O
*
NH2
n
Poly-L-Ornithine
H2N CH COOH
(CH2)2
NH
C O
O
CH2
N-Carbobenzyloxy-L-Ornithine
ClCCCl3
O
diphosgene
EtOAc DCM/EtOAC (2/1)
Bu3N
CHCl3
HBr/CH3COOH
Continued Synthesis
pag. 7 V. Vermeersch – PolExGene Prague – 22/05/2008
Conversion into Poly-L-Arginine: specific ‘guanidylating’ agent (97%)
* NH CH C
(CH2)3
O
*
NH
n
NHH2N
N
N
HN NH2
* NH CH C
(CH2)3
O
*
NH2
n
+
(pH = 9.5)
KI (6 M)
H2ON
HN
+
Polyornithine 3,5-dimethyl-1-guanylpyrrazole
Polyarginine 3,5-dimethylpyrrazole
Characterisation
- Mv (poly-N-CBZ-Ornithine) in CHCl2COOH
- NMR & IR spectra
- Mw (Gel Permeation Chromatography)
Method of analysis currently optimised (GPC detector down)
Range of M/I ratio’s
5/1 – 10/1 – 25/1 – 100/1 – 250/1
Mw = ?
GMBS
Intermediate: GMBS coupling
Peptide Coupling
pag. 8
(CH2)2
C O
NH
CHNH C*
O
m
CH2
N
H3C CH3
2
(CH2)2
C O
NH
CHNH C
O
CH2
NH2
2
n-x
C O
(CH2)3
NOO
(CH2)2
C O
NH
CHNH C *
O
CH2
NH
2
x
SCH2
Penetratine (CPP)
Ac
CPP-coupled polymer
pDMAEG-AEG-CPP (V09/V10)
V. Vermeersch – PolExGene Prague – 22/05/2008
NH2-polymer
Thiol-coupling
Thiolated CPP’s (Montpellier)
Penetratine (available) V09 (1,65% & 2,79%) V10 (1,77%) PEI (0,47%)
Octa-Arginine No spacer CH2-moiety spacer (3/5/7) PEG spacer (2/11)
RGD CycloRGD
Physico-chemical testing
Agarose Gel-electrophoresis: Condensation point V11-V12-V13
(in nm) CR 0h 48h
V13a
1-1 158* 466*
2-1 32 42
4-1 34 46
V12 V11
DNA DNA
CR 1.0
CR 1,8
V13a
Dynamic Light Scattering (DLS)
(in nm) CR 0h 48h
V13b
1-1 57 400
2-1 30 57
4-1 30 67
0,0
20,0
40,0
60,0
80,0
100,0
120,0
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 2,2
V13a
V13b
EtBr Fluorescence
pag. 9 V. Vermeersch – PolExGene Prague – 22/05/2008
Physico-chemical testing
Time (s)
Flu
ore
scen
ce y
ield
Single-shot kinetics (2-1 CR)
pag. 10 V. Vermeersch – PolExGene Prague – 22/05/2008
Future plans
Synthesis of combined polymers - Guanidine-Glutamine - Guanidine-PEI - Guanidylated PLL
Coupling of Octa-Arginine - Polyglutamine copolymers (V05, V06) - Polyarginine (V13) - PEI - CPP density variation
Zeta-potential measurments of V09-V13 (+ V13 derivatives) DLS of CPP-coupled polymers
(Polymers with free –NH2)
pag. 11 V. Vermeersch – PolExGene Prague – 22/05/2008
PBM products sent-out
GROUP V13a/b VT01-02-03-04 VT07 2/3/4 VT09/10
ENS x/x x/x/x/x x/x/x x/x
UH.FP x/x x/x/-/- x/-/- x/x
IBMT -/x x/x/-/- x/-
UKU
ARK
UAT
IMIC -/x x/x/-/- x/-
CNRS
pag. 12 V. Vermeersch – PolExGene Prague – 22/05/2008