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

Targets for plasmatic coagulation inhibition

Thrombin: center molecule

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.