Methods of Biomaterials Testing - Blood and Vessel
Transcript of Methods of Biomaterials Testing - Blood and Vessel
Design of hemocompatible surfaces
actively
•
Hemocompatibility – basic outline (repetition)
•
Molecules and strategy for active inhibitory surfaces
•
Basics on surface modification
•
Examples of research projects and results
blood
biomaterialinteraction
blood
biomaterialinteraction
First reactions on biomaterials surfaces
CH3NH2OH2CH2CH2
CH3NH2OH2CH2CH2
CH3NH2OH2CH2CH2
CH3NH2OH2CH2CH2
protein adsorptionsurface properties
enzyme activation
cell adhesion, cell activation
Interaction of blood with biomaterial surfaces depending on:
blood coagulation
inflammatory reactions
Our aimspecific anticoagulant reactions directly on material surface
less adverse effects leading to non functionality of biomedical product
lowered systemic anti-coagulation
Werner C, Maitz MF, Sperling C. Current
strategies
towards
hemocompatible coatings. J Mater Chem
2007;17:3376-84.
Modification strategiesmodification of materials for passivation
immobilization of specific inhibitors to stopp coagulation processes
biomimeticnatural synthetic
Inhibitors for immobilization
HeparinThrombomodulin
Protein CNitric oxide NO
Modified glucose and maltose
Sulfonated cellulose
Benzamidin- derivatives
PPACK
Inhibition sites on thrombin
Direct inhibitors of thrombinNatural inhibitor: heparin, thrombomodulin, hirudin
Biomimetic inhibitor : modified glucose and maltose, sulfonated cellulose
Synthetic inhibitor: benzamidinderivatives, PPACK
PP INHIBITOR
INHIBITOR
Specific activityResistance to proteolytic enzymesStructural stabilityFacilitated processing
ALKYL CHAINPOLYETHYLENE GLYCOL
SPACERS
Better biocompatibilityBetter solubility in aqueous solvantLack of toxicity and immunogenicity
P = Maleic anhydride copolymers
POLYMERIC SUPPORT
O OO
CHCH2
R
n
Regular alternating reproductiblestructure
BiocompatibilityVaried hydrophobicityReactivity of anhydride group
Strategy of surface immobilization
Pompe T, Zschoche
S, Herold N, Salchert
K, Gouzy
MF, Sperling C, Werner C. Maleic anhydride
copolymers-a
versatile
platform
for
molecular
biosurface
engineering. Biomacromolecules. 2003;4:1072-79.
Polymeric supports –
Preparation of the PO-MA films
OH
Si OO
O
CH CH
O
O
Si OO
O
SiCH3 CH3
CHCH
N OO
CH CH2CH2CH
OO
R1 R1n
Spin coatingor
solution
casting
H2N
HNNH2CH2CN
H
O
11
R2 =R1 = (CH2 )15 CH3
nnn))) (( (+ R2NH2 CH2 CH
R1
CH CH
C CN
OO
R2
CH2 CH
R1
CH CH
C C
O
OO
CH2 CH
R1
CH CH
C C OO
NHOH
R2
2-4h/120°C
Conversion
CH CH
N
O
Si OO
O
SiCH3 CH3
CHCH
N OO
CH CH2CH2CH
OO
R1R1
R2
n
3 aminopropyldimethylethoxysilane
120 °C, 2 hours
O
Si OO
O
SiCH3 CH3
H2N
Strategy of surface immobilization
Chemical surface characterization
QuantityEllipsometry - layer thickness
XPS – quantification of specific atomic composition
Quantification of immobilized protein (HPLC of hydrolyzed protein)
QualityFluorescence labeling antibodies to inhibitor
bound thrombin (to inhibitor)
Activity assay (of immobilized enzymes or of enzymes after interaction with immobilized inhibitor)
In vitro hemocompatibility assay
Incubation with freshly drawn whole blood•
Samples: top / bottom of round chamber, sides PTFE
•
Anticoagulation with heparin (2 IU / ml)•
incubation with slow rotation for 2 h at 37°C
Parameters for hemocompatibility assessment
Plasma (ELISA) TAT, C5a, PF4, Elastase, TF
Blood numbers of leukocytes, plateletscell activation (CD 11b)conjugates of LZ / plt
Material surface adherent cells, fibrin formation motor
rotation
rotation
Epicyclic gear
Streller
U, Sperling C, et al. J Biomed
Mater Res 2003;66B:379-90.
Heparin as an anticoagulant
Heparin: polysaccharide – glycosaminoglycane
mass
between
4.000 and 40.000 (peaks
at appr. 15,000)
low
molecular
weight
heparins: molar
mass
at 5,000.
Natural form: Heparan sulfate
O
O
OH
CH2OSO3
HN
O
O
OH
COOH
OH
OH
CH3
O
OOSO3
CH2OH
NH
OOH
COOH
OSO3
O
O
SO3
OOH
CH2OSO3
NH
O
•
found in all animal tissues as a proteoglycan
•
binds to a variety of protein ligands
•
regulates a wide variety of biological activities•
developmental processes
•
Angiogenesis•
blood coagulation
•
tumour
metastasis
Heparin as an anticoagulant
Di Nisio, N Engl
J Med 353, 1028 (2005)Anticoagulant effect
heparin
binds
to antithrombin III which
binds
thrombin
1,000-times faster.
Inhibition of thrombin, FX, FXI, FIX and FXII.
Immobilized
heparin
releases
ATIII/heparin
complex
and is
ready
for binding
of more
ATIII.
Immobilization of heparin
Surfaces: Polyethersulfon
PES coated
with
albumin and heparin multilayers
in layer-by-layer
technique:
sequential adsorption of protein (albumin) and polyelectrolyte (heparin) on a surface under conditions where the constituents bear opposite net charges.
Samples: PES
Albumin, bovine (BSA)
Heparin (HEP), standard heparin
Endurin
(END), high anticoagulant active fraction of heparin
Test system: perfusion systempump
Incubation chamber
fill intake out
pump
Incubation chamber
fill intake out
Heparin
PES BSA HEP END
103
104
105
plat
elet
s / m
m2
IV PES BSA HEP END10
1001000
4000
20003000
5000
PF4
[IU/m
l]
IV PES BSA HEP END
1000
2000
40003000
5000
500
TAT
[µg/
l]
IV PES BSA HEP END
10
40
30
20C
5a [µ
g/l]
Heparin
PES
BSA
HEP
END
ResultsHemocompatibility enhanced
Results less stringent than expected
Problems !!
Scientific reason for positive effect not fully understood
Sterilizability
Storage stability
Stability in vivo
Sperling C, Houska
M, Brynda
E, Streller
U, Werner C. In vitro hemocompatibility of albumin-heparin
multilayer
coatings
on polyethersulfone
prepared
by
the
layer-by-layer technique. J Biomed
Mater Res A 2006;76:681-89.
Thrombomodulin (TM)
•
Binds
thrombin
and inactivates
the
procoagulant properties•
Activates
anticoagulant
properties
of thrombin: Activation
of protein
C
–
Inactivation
of FVa
and FVIIIa
•
Antiinflammatory
effects–
Protein C/endothelium
–
lectin-like
domain
Esmon
2003, Chest
124, 26S
Major anticoagulant
molecule of the
blood
vessel
wall
0.0
0.5
1.0
1.5
2.0
0 2 4 6t [min]
OD
405
nm
TM Jeffamin
Activity assay of immobilized thrombomodulin
TMTMTM TM
TMTM
TM TM
PEG
PEG-TM
PEG
PC APC
TM
peptide+pNA
substrate
S2366
IV G P TM BD0
1000
2000
3000
4000
5000
TAT
[µg/
l]Activation of coagulation on tested surfaces
IV Initial valueG GlassP PTFETM
Thrombomodulin
BD Benzamidinderivative(direct
thrombin
inhibitor)
Reference: PP-MSA (base
material)
TAT base layer PP-MSA
5429 ± 361
CH CHO
OSi OOO
SiCH3 CH3
CHCHN OO
CH CH2CH2CH
OO
CH2 nCH2
IV G P TM BD0
1000
2000
3000
4000
PF4
[IU/m
l]
G P TM BD50
60
70
80
90
100
Plat
elet
s [%
IV]
Activation of platelets on tested surfaces
IV Initial valueG GlassP PTFETM
Thrombomodulin
BD Benzamidinderivative(direct
thrombin
inhibitor)
PF4 PP-MSA
2416 ± 209
Platelets PP-MSA
60 ± 15
glass PTFE TM
Leukocyte adhesion and activation
IV glass PTFE PEG PEG-TM0
500
1000
CD
11b
[a.u
.]
IV G P TM BD012345
C5a
[µg/
l]
ResultsHemocompatibility is enhanced significantly
Problems !!High price
Storage stability
Stability in vivo
Conclusion
Sperling C., Salchert
K., Streller
U., Werner C., Covalently
immobilized
thrombomodulin inhibits
coagulation
and complement
activation of artificial
surfaces
in vitro. Biomaterials 2004; 25: 5101-5113.
Activated Protein C
•
Serine
proteasis•
62 kDa
•
Incativates
coagulation factors
FVa
and FVIIIa
•
Clinically
applied
(Xigris®)•
Half time plasma: appr. 15 min –
Protein C Inhibitor PCI-1, PCI-2
–
α1 proteinase
inhibitor–
α2 macroglobulin
–
PAI
Murakami
M.T. (2005) J Biol
Chem
280, 39309
Prothrombin Thrombin
Factor
XaFactor
X
F IXa
FVa
FVIIIaPhospholipids
Copperhead Snake Venom Protac®
Venom
of the
snake Agkistrodon contortrix
contortrix•
Rapid, cofactor-independent
activator
of protein
C•
Single chain, glycosylated
serine
protease•
37 kDa, IP 3.0
•
No physiological
inhibitor/regulation
Murakami
M.T. (2005) J Biol
Chem
280, 39309
•
In vivo applications
not
published•
Sensitation/ allergic
reactions
•
(sensitive to benzamidine
inhibitors)•
Expenses: 50€/3 Units
Coagulation activation of surfaces
0.1
1.0
10.0
100.0
Glass PTFE Xigris PP-MA BSA
TAT
rela
tive
to P
TFE
0
1
2
3
4
Glass PTFE Xigris PP-MA BSA
C5a
rela
tive
to P
TFE
0
1
10
Glass PTFE Xigris PP-MA BSA
PF4
rela
tive
to P
TFE
ResultsHemocompatibility only slightly enhanced
Problems !!Sterilizability
Storage stability
Stability in vivo
Nitric oxide eluting polymers
Nitric oxide – NOFree radical gas
Mainly formed in endothelial cells (also platelets, erythrocytes,…)
NO diffuses to smooth muscle cells and induces vasodilation, regulates blood pressure
Also important anti-platelet
anti-oxidant
anti-adhesive
anti-proliferative
Inhibits platelet aggregation
platelet activation
BUT: very short half-life!
NO is produced from the amino acid L-arginine
by the enzymatic action of nitric oxide synthase
(NOS).
Nitric oxide eluting polymers
PVA NO
PVA
PSPE NO
PSPE
PTFE
glass
100 1000 2000500
TAT [µg/l]
PVA NO
PVA
PSPE NO
PSPE
PTFE
glass
10 100 1000 2000
PF4 [IU/ml]
Leukocyte Thrombocyte Conjugates
Conjugates [% Leukocytes]0 20 40 60 80 100 120
Glass
PTFE
PSPE native
PSPE NO
PVA native
PVA NO
GranulocytesMonocytes
Cooperation with Prof. Marcelo de Oliveira from Campinas / Brazil
PSPE: poly(sulphydrylated polyester) / poly(methylmetacrylate) PSPE/PMMANitrosation
reaction in HCl
aqueous solution containing 0.40 g of sodium nitrite (NaNO2) for 15 minutes at room temperature.
PVA: Poly (vinyl
alcohol) (PVA) films
partly
already
containing
S-nitrosoglutathione
(GSNO
Nitric oxide
ResultsHemocompatibility enhanced for hydrophobic polymer for platelet related parameter
Preparation of materials still problematic
Problems !!Sterilizability
Storage stability – preparation right before use?
Stability in vivo – very short half life
Modified polysaccharides
Grombe
R., Gouzy
M.F., Maitz
M.F., Freundenberg
U., Zschoche
S., Simon F., Pompe T., Sperling C., Werner C., Sulfated
glycopolymer
thin
films
-
preparation, characterization
and biological
activity. Macromol
Biosci
2007; 7: 195-200.
Antithrombin binding capacity
Antithrombin III binding follows the row:
Heparin > sulf. polysacharide > sulf. disasscharides > sulf. monosaccharides > negative surface charge
ResultsHemocompatibility not enhanced sufficiently with surfaces tested so far
H2N
H2NNH
NH3
O 7
specific benzamidine based synthetic thrombin inhibitor 1 (BA)
Interaction of synthetic inhibitor with thrombin
Binding pocket of thrombin with benzamidine in active site
Ki
benzamidine: 779 ±
160 µM
Ki
BA synthetic
inhibitor
1: 103 ±
10 µM
Interaction of synthetic inhibitor with thrombin
Binding pocket of thrombin with benzamidine base synthetic thrombin inhibitor 2 in active site
(CH2)4
SNC
NCN
O
OHO
HO
C
NH2
NHH2N
Ki
benzamidine: 779 ±
160 µM
Ki
BA synthetic
inhibitor
1: 103 ±
10 µM
Ki
BA synthetic
inhibitor
2: 0.064 ±
0.005 µM
Anti-thrombin activity
y = 192x + 0,658R 2 = 0,993
y = 68,0x + 0,689R 2 = 0,999
y = 274x + 0,709R 2 = 0,999
y = 6,54x + 0,656R 2 = 0,881
0
2
4
6
0,000 0,005 0,010 0,015 0,020 0,025 1/S [µM-1]
1/v
[min
x μ
M pN
A]
n I 1000 µM 2500 µM 5000 µM
Hydrolysis of the S-2238 chromogenic substrate by thrombinin the presence and absence of TI 2 (Lineweaver-Burk plot)
Ki determination
p-Aminobenzamidine 779 ± 160 µM
Thrombin Inhibitor TI 2 83 ± 17 µM
NH2
HN
H2N
H2N
H N
NH2CH2CNH
O
1 1
p-Aminobenzamidin
TI 2
NH
C
CH2
O
N
HN NH2
(CH2)11
O
NH2
TI 3Thrombin Inhibitor TI 3 16 ± 7 µM
PO hyd PO Jeff PO TI PO-PEG TI0,25
0,30
0,35
0,40
OD
[405
nm
]
Thrombin inhibition by modified surface(incubation for 60 min at RT with substrate S 2238)
Modified interfaces:
hydrophobicity
and anti-thrombin activity
O OO
PO-MA
O OH O OH
PO-MA
POhyd
O N O
O
(CH2CH2O)nO
CH3
PO-MA
POJeff
H2N NH
O N O
PO-MA
POTI
O N O
H2N NH
HN
PEG600
HN
O
O
PO-MA
PO-PEGTI
Dynamic contact anglesliquid=millipore
water; velocity= 0.5 µL/s
Contactangle (°)
POhyd POJeff POTI POPEGTI
Adv. 100.7± 4.0 95.9±1.0 94.7±1.6 88.0±1.0
Rec 60.0±4.0 41.9±1.0 52.1±1.4 26.5±1.5
Immobilisation of thrombin inhibitors
Thrombin-Antithrombin Complex
COAGULATION
Platelet Factor 4
THROMBOGENICITY
Complement 5a
INFLAMMATION( immune response)
0
10
20
30
40
C5a
[µg/
l]
0
50
100
150
200
PF4
[IU
/ml]
0
20
40
60
80
800
1000
TAT
[µg/
l]
I.V. POhyd POJeff POTI PO-PEGTI I.V. POhyd POJeff POTI PO-PEGTI I.V. POhyd POJeff POTI PO-PEGTI
PTFE TAT = 91.5 +/- 11.5 μg/L
PTFE C5 a = 2.5 +/- 0.5 μg/L
PTFE PF 4 = 87+/- 5 IU/mL
I.V. POhyd POjeff POTI PO-PEGTI
Platelets *109/l 293 225 261 266 261
SD 4.5 93.1 18.0 37.8 18.3
Hemocompatibility of thrombin inhibitors
POhyd = Hydrolyzed
PO-MA
POJeff = PO-MA + JeffaminePO-PEGTI = PO-MA + PEG spacer
+
TI
POTI = PO-MA + TI
Hemocompatibility of thrombin inhibitors
Scanning electron microscopy after blood incubation
Gouzy
M.-F., Sperling C., Salchert
K., et al., Biomaterials 2004; 25: 3493-3501.
Gouzy
M.-F., Sperling C., Salchert
K., Pompe T., Rauwolf C., Werner C., Biointerphases
2006; 1: 146-155.
ResultsHemocompatibility enhanced
Problems !!Stability in vivo
Saturation effects
PPACK
•
Peptidic
structure: D-Phe-Pro-Arg-chloromethylketon•
Inactivates
coagulation
factor
thrombin•
Irreversible inhibitor
(1:1 complex
with
thrombin)•
Inhibitor constant Ki=0.1 µM
NH2
O
NH
N
O
Cl
O
NH2N
NH2
Thrombin
N-PP
ACK
Thrombin
fibrin
ogen Fibrin +
Clot
formationfib
rinog
en
fibrinogen
PPMA copolymer
O N O
O
(CH2CH2O)nO
CH3
O N O
O
(CH2CH2O)nO
CH3
PPMA copolymer
O N O
H2N NH
HN
PEG600
HN
O
O
O N O
H2N NH
HN
PEG600
HN
O
O
Thrombin
N-PP
ACK
PPMA copolymer
O N O
H2N NH
HN
PEG600
HN
O
O
O N O
H2N NH
HN
PEG600
HN
O
O
ThrombinN-
PPAC
K
Thrombin Thrombin
PP-MA PPACK aPCPPMA copolymer
O N O
H2N NH
HN
PEG600
HN
O
O
O N O
H2N NH
HN
PEG600
HN
O
O
BSA BSA
BSA
Thrombin
TAT: activation
of coagulation
enzymes
PF4: activation
of blood platelets
PPACK
• Successful immobilization and surface characterization
• Improved hemocompatibility parameters for inhibitor surfaces
• Effects less concise than expected
• Saturation effects!
PPACK
PP-MA aPC PPACK BSA
1
10
543
TAT PF4
activ
atio
n re
lativ
e to
aPC
2
Take home message
Immobilisation
strategy
mostly
targets
plasmatic
coagulation.
Versatile
molecules
exist
for
surface
immobilization:naturalbiomimeticsynthetic
Synthetic
molecules
less
cost
intensive, less
problems
with storage
as well as in vivo stability
and sterilizability
Complex
in vivo situation
–
no fully
satisfying
surface modulator
found
yet
Only
heparin
immobilization
as industrial
product
up to now.