Attachment of ADH Modified Heparin onto Silica Wafers

1. 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

2. 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

3. Cleaning Silica Wafers • 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 Above is a magnified picture of the surface of a silica wafer covered in dust

4. 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

5. ADH Modification of Heparin • This process changed the heparin so that it was both internally-modified 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}-3-ethylcarbodiimide hydrochloride and placed in dialysis tubing for 4 days

6. 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

7. 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

8. 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 anti-coagulant then the un-modified heparin

9. ContactAngles • 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