Transcript Preparation Anti-Thrombotic Materials For Blood Contact

Attachment of ADH
Modified Heparin onto Silica
Wafers
By Amy Mayberry
& Jonathan McGrath
Mentors: Brianna Anderson-Gregg, Hyo Jin, and Omkar
Oregon State University, Department of Chemical Engineering
Introduction
• When an injury occurs to the endothelial cells in the tissue the
coagulation cascade begins in which a series of proteins ending
with thrombin are triggered and a clot is created
• Heparin is a commonly used anti-coagulant that works by
allowing antithrombin III to inactivate the thrombin protein and
other proteins needed for the blood to coagulate
• By modifying the heparin we hope to be able to make a form of
heparin that can attach to the biomaterials used in the body and
still act as an anti-coagulant to prevent clots from forming and
sticking to the biomaterials
Cleaning Silica Wafers
Above is a magnified picture of the
surface of a silica wafer covered in dust
• Particles of dust or other
chemicals can alter the
process of heparin
attachment
• It does this because
hydrophobic dust particles
on the coated silica wafer’s
hydrophilic surface inhibit
the cohesion of water
• To insure that the silica
wafer’s surface is as clean as
possible it needs to undergo
an extensive cleaning process
Silanization of Silica Wafers
• The silanization process aminates the silica wafers with
Aminopropyltriethoxy silane where the –OH groups on the
surface of the untreated silica wafers are replaced by -NH2 and
the surface of the wafers becomes hydrophobic
• During the procedure the cleaned silica wafers were placed in a
2% solution of APTS and stored for 24 hours at 50° C
Si-OH + Aminopropyltriethoxy silane  Si-NH2
ADH Modification of Heparin
• This process changed the heparin so that it was both internallymodified and end-modified and contained a –NH2 , causing it to
become hydrophobic
• ADH, or Adipic dihydrazide, was used to modify 12,500 Dalton
Heparin
• The unfractionated heparin was mixed with ADH (Adipic
dihydrazide) and EDCI (1-{3-(Dimethylamino)propyl}-3ethylcarbodiimide hydrochloride and placed in dialysis tubing for
4 days
Carboxylation of Silica Wafers
• This process used succinic anhydride to carboxylate the aminated
silica wafers and the –NH2 groups were replaced by –COOH
groups on the silica, causing it to become strongly hydrophobic
• The aminated silica wafers were placed in a solution of succinic
anhydride for 10 hours
Si-NH2 + succinic anhydride  Si-COOH
Attachment of ADH Heparin to
Silica Wafers
• In this process the silica wafers were placed in a mixture of the
ADH modified heparin, EDCI, and bis-tris HCL for 24 hours
• Both the silica wafers and the modified heparin had high
hydrophobicity which bonded them together and attached the
ADH heparin to the silica wafers
APTT Coagulation Analyzer
• To measure the time for the ADH modified heparin to coagulate
we used horse plasma, APTT, and a sample of the heparin in a
coagulation analyzer
• We found that the ADH modified heparin took less time to
coagulate the horse plasma then the un-modified heparin did,
meaning the ADH modified heparin was a less effective anticoagulant then the un-modified heparin
Contact Angles
• Contact angles were used to measure the degree of
hydrophobicity of the coated silica wafers
• In this process a drop of water is placed on a silica wafer and a
computer takes a picture of the wafer’s surface. The computer
then measured the degree of the angle of the water droplet’s
base and top
• If the silica wafers were well coated with ADH heparin they
would be hydrophilic and therefore have a contact angle of
around 25º
• We found that one set of silica wafers was in this range and had
been well coated with the ADH heparin while the second set of
wafers had larger contact angles which could mean they had not
been fully coated with the ADH heparin or that dust on the
surface of the wafers had repelled the water and altered the
results