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Fibrinogen Adsorption on Antimicrobial Modified Surfaces

Fibrinogen Adsorption on Antimicrobial Modified Surfaces. Julie Auxier Dr. Joseph McGuire, Bioengineering Oregon State University HHMI 2009. Imperfect Implants. Problems from implanted devices: Clotting; embolism risk Bacterial adhesion; infection Overall implant rejection

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Fibrinogen Adsorption on Antimicrobial Modified Surfaces

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  1. Fibrinogen Adsorption on Antimicrobial Modified Surfaces Julie Auxier Dr. Joseph McGuire, Bioengineering Oregon State University HHMI 2009

  2. Imperfect Implants • Problems from implanted devices: • Clotting; embolism risk • Bacterial adhesion; infection • Overall implant rejection • Treat with heparin and other anticoagulants • Risk of platelet depletion, excessive bleeding

  3. Thrombosis and Blood Proteins • Thrombosis: formation of a blood clot in a blood vessel which obstructs blood flow Common Pathway Factor X Prothrombinase Factor VII Tissue Factor Complex Factor X Activator Complex Clotting Factor VII Clotting Factors VIII, IX Thrombin Prothrombin Ca2+ Ca2+ Fibrinogen Fibrin Platelet Factor PF-3 Tissue Factor III Activated Proenzymes, usually Factor XIII Extrinsic Pathway Tissue Damage Intrinsic Pathway

  4. Thrombosis and Blood Proteins • Fibrin forms the scaffolding, platelets fill the holes • Late stent thrombosis possibly caused by: • Early discontinuation of anticoagulant medication • Stent fracture • Abnormal reaction of tissue to implant material • Small lumen size, slow flow rate

  5. Prevention with Pluronic® F108 F108 approximate maximum length: 50nm Approximate length of a red blood cell: 5µm (500nm) HYDROPHILIC HYDROPHOBIC PEO PEO HYDROPHOBIC SURFACE PPO

  6. How Brush Layer Functions BACTERIA PROTEIN HYDROPHOBIC

  7. Hydrophobic Surface Nisin - Lantibiotic • Inactivate bacteria by creating a pore and destabilizing the membrane • Naturally made from bacteria Lactococcuslactis • Used in food products: preservative, making cheese • No evidence suggests nisin induces an immunogenic reaction (based on previous studies)

  8. Previous Research • Change between pluronic coating with nisin before and after challenged with fibrinogen. • Two possibilities may account for the lower signal

  9. Purpose Identify fibrinogen adsorption on non-fouling, antimicrobial surfaces.

  10. Hypothesis The pluronic layer maintains its protein repelling nature despite nisin loading. Hence, fibrinogen more likely will not adsorb to the surface and will displace nisin when repelled.

  11. Methodology • Surface preparation: • Silanize silica to make surface hydrophobic • Covalently attach pluronic F108 by gamma radiation • Load brush layer with nisin • Protein assay tests (ELISA) • FITC labeling fibrinogen • Parallel flow platelet adhesion tests Inactivated Platelets Fibrinogen Surface

  12. Enzyme Linked Immunosorbant Assay Block well with bovine serum albumin (BSA) or milk. “Tagged” fibrinogen antibody detects fibrinogen in a sample, and then a colorimetric substrate detects the antibody Fibrinogen sticks to sample surface. Add enzyme-linked antibody which attaches to fibrinogen. Add colorimetric substrate to react with enzyme on antibody. Surface Solution changes color, read absorbance at 490nm.

  13. Results *Not to scale.

  14. FITC labeling fibrinogen Fluoroscein isothiocyanate reacts with N-terminal amines on fibrinogen. Prepare labeled fibrinogen solution. Contact surfaces (microspheres) with labeled fibrinogen. Rinse thoroughly. Dissolve microspheres with NaOH. Read absorbance at 490nm.

  15. Results

  16. Parallel Flow Platelet Adhesion Flow chamber allows for evenly distributed flow at a constant rate (16 mL/min, shear rate 480 sec-1) Flow platelet-rich equine plasma through system. Buffer wash. Fix platelets with gluteraldehyde. Buffer wash. Dehydrate with ethanol. Critical point dry. Image with SEM.

  17. Results

  18. Conclusions • ELISA and FITC-Fg results indicate: • Brush layer effectively inhibits fibrinogen adsorption. • Addition of nisin to the brush layer does not promote fibrinogen adsorption. • Platelet adhesion studies require refining before definitive results may be collected.

  19. Future Work • Continue work with parallel flow chamber. • Repeat FITC-Fg tests. • Investigate labeling fibrinogen with trifluoroacetic anhydride which can be quantified using x-ray photon spectroscopy (XPS).

  20. Acknowledgements Great deal of thanks to: • Dr. Joe McGuire • Karl “Rat” Schilke • Dr. Karyn Bird • Matt Ryder • Lars Bowlin • Howard Hughes Medical Institute • Allvivo Vascular Inc.

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