ugent contribution to polexgene (wp 3 & wp 4) polexgene technical meeting prague 22-23/05/2008...
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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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 3
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)
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 4
• 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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 5
• 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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 6
PI grafted with gelatin type A
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 7
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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 8
• 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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 9
PI
PI + gel-MOD
1 µm
Coating of gold chips with CIP’s/CPP’s
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 10
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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 11
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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 12
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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 13
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)
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 14
• 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)
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 15
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
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 16
PBM products sent-out
S. Van Vlierberghe – PolExGene Prague - 22/05/2008 pag. 17
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