ugent contribution to polexgene (wp 3 & wp 4) polexgene technical meeting prague 22-23/05/2008...

UGent contribution to PolExGene(WP 3 & WP 4)

Polexgene Technical meetingPrague 22-23/05/2008

PBM-GGentent

UU

Polymer Chemistry & Biomaterials GroupPolymer Chemistry & Biomaterials Group

S. Van Vlierberghe, V. Vermeersch,

P. Dubruel & E. Schacht

Overview

• Workpackage contents

• Summary of last presentation

• Planning since previous meeting

• Work Performed

• Ongoing & future plans

S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 2

Workpackage Content


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

Workpackage 4:Preparation of plasmids and CPP-containing polyplexes

Study of the coating of polymer membranes with CPP-containing polyplexes

Summary Ghent meeting (Dec 2007)


• PI grafted with gelatin type B1. Aminopropylmethacrylamide + gel-MOD

2. Succinimidylester of AFB + gelatin

• Characterization of grafted PI1. ATR-IR spectroscopy

2. Atomic force microscopy (AFM)

3. X-ray photo-electron spectroscopy (XPS)

4. Static contact angle measurements (SCA)

• Preparation of polymer membranes based on gelatin type A

(IEP = 8) (IEPgelatin B = 5).

• Preparation of polymer membranes based on gel-MOD + HA-MOD (1 – 5 w/v%) gel-MOD + CS-MOD (1 – 5 w/v%)

• SPR measurements:Study of interaction between gel-MOD (A, B) and polyplexes.

• WP 4: Coating of PI membranes with CPP-containing polyplexes.

• Coating of gold chips with CPP’s and CIP’s → impedance• Modification of gelatin with DTPA (to chelate technetium)• Coating of bare stents with gelatin-DTPA and gelatin + glycerol

Planning since previous meeting


PI grafted with gelatin type A


O

O

N

O

O

N

n

O

NH

H2N+

O

O

HN

O

O

NH

n

NH

HNO

HN

HNO

Gel NH2 + H2C C

CH3

C

O

O C

O

C

CH3

CH2 Gel NH C

O

C

CH2

CH3

+ Gel Gel

Gel

Gel+ Irgacure + UV

PI

aminopropylmethacrylamide

gel-MOD (type A)

MeOH, 21°C

5 w/v%

gel (type A)

Characterization of PI + gel-MOD A


• ATR-IR spectroscopy• Gelatin A characteristic peaks: 1629 cm-1, 1538 cm-1

• IR-peaks (PI + gel-MOD type A): 1622 cm-1, 1547 cm-1

50

60

70

80

90

100

110

1000150020002500300035004000

Wavenumber (cm-1)

% T

ran

smit

tan

ce

Gel-MOD (type A)PI + gel-MOD A

Porous PI


PI

PI + gel-MOD

1 µm

Coating of gold chips with CIP’s/CPP’s


Cyclo-RGD: 1634 cm-1, 1544 cm-1

Au + cyclo-RGD: 1663 cm-1, 1545 cm-1

RGD: 1640 cm-1, 1528 cm-1

Au + RGD: 1680 cm-1, 1541 cm-1

Pen-SH: 1648 cm-1, 1531 cm-1

Au + pen-SH: 1652 cm-1, 1558 cm-1

95

95,5

96

96,5

97

97,5

98

800130018002300280033003800

Wavenumber (cm-1)

%T

ran

smit

tan

ce

Au + pen-SH

93,9

94,4

94,9

95,4

95,9

96,4

96,9

800130018002300280033003800

Wavenumber (cm-1)

%T

ran

smit

tan

ce

Au + cyclo-RGDAu + RGD

Atomic composition (%) sample Au C N O Ga

Au 8 62 - 14 15 Au + cyclo-RGD 10 49 6 14 21

Au + RGD 5 66 4 15 11 Au + penetratin 8 56 5 14 17

→ chemisorption (10 mM)

Gelatin modification with DTPA


Chelation of Gd or Tc

=> visualization of gelatin coating on stents

N

NH

N

O

O

O

O

O

O

O

O

Gel NH2 +

GelNH

O

N

O

O

NH

O

O

N

O

OO

O

gelatindiethylenetriaminepenta-acetic acid dianhydride

gelatin-DTPA

pH 7, 37°C

Characterization using IR-spectroscopy


IR-spectrum of gelatin-DTPA: 1091 cm-1 and 1630 cm-1

=> coupling of DTPA to gelatin

30

40

50

60

70

80

90

100

110

700120017002200270032003700

Wavenumber (cm-1)

%T

ran

smit

tan

ce

GelatinGel-DTPA

1H-NMR spectroscopy


N-CH2-COO-

Val, Leu, Ile

Gel-DTPA I3.5 ppm 294 I1.2 ppm 1000

DS (%) = 0.3836 mol x [ (I3.5 ppm / 10) / I1.2 ppm] x (100 / 0.0385 mol)

5% → 30%

DS (gel-MOD) = 65%

PEI

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

-100 100 300 500 700 900 1100 1300 1500

Time (s)

Res

po

nse

(R

U)

2:14:18:11:1

Gelatin affinity for polyplexes (SPR)


• Gelatin B V03

-2000

0

2000

4000

6000

8000

10000

12000

-100 100 300 500 700 900 1100

Time (s)

Res

po

nse

(R

U)

1:12:14:1

V01

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

-200 300 800 1300 1800

Time (s)

Res

po

nse

(R

U)

2:14:18:11:1

V01 (2:1)

-500

0

500

1000

1500

2000

2500

3000

0 50 100 150 200 250 300 350

Time (s)

Res

po

nse

(R

U)

5 µl10 µl25 µl50 µl100 µl

Gelatin affinity for polyplexes (SPR)


Gelatin A (+) Gelatin B (-)V03

-100

0

100

200

300

400

500

600

700

-200 0 200 400 600 800 1000 1200 1400

Time (s)

Res

po

nse

(R

U)

1:12:1

V03

-2000

0

2000

4000

6000

8000

10000

12000

-100 100 300 500 700 900 1100

Time (s)

Res

po

nse

(R

U)

1:12:14:1

lower affinity for polyplexes (+)

higher affinity for polyplexes (+)

• Gelatin immobilization on porous PI => open pores?• Reduce sample size of PI to 0.5 mm for implantation in mice • Preparation of polymer membranes based on

gel-MOD + HA-MOD (1 – 5 w/v%) gel-MOD + CS-MOD (1 – 5 w/v%)

• WP 4: Coating of PI membranes with CPP-containing polyplexes=> Qualitative + quantitative analysis

• Coating of bare stents with gelatin-DTPA and gelatin + glycerol• Modification of biodegradable membranes with gelatin

(Vivosorb®) → feasibility study

Ongoing and future plans


PBM products sent-out


GROUPPI +

gel-MOD A/Bgel-MOD A/B

Irgacureglass +

gel-MOD B wp +

gel-MOD A/BPI + //

PI + AFB

ENS

UH.FP X

IBMT

UKU

ARK

UAT X / -

IMIC - / X X / X / X X X / X X / X

CNRS

ugent contribution to polexgene (wp 3 & wp 4) polexgene technical meeting prague 22-23/05/2008...

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