Attachment of ADH Modified Heparin onto Silica Wafers

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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
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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

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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 wafers hydrophilic surface
  inhibit the cohesion of water
• To insure that the silica wafers 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
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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
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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

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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

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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

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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

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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 wafers surface. The computer then measured
  the degree of the angle of the water droplets
  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