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