Transcript Fibrinogen Adsorption on Antimicrobial Modified Surfaces Synthesis

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
 Treat with heparin and other
anticoagulants
 Risk of platelet depletion, excessive
bleeding
Thrombosis and Blood Proteins
 Thrombosis: formation of a blood clot in a blood vessel which
obstructs blood flow
Common Pathway
Factor X
Factor VII Tissue
Factor Complex
Factor X Activator
Complex
Prothrombinase
Clotting
Factor VII
Clotting Factors
VIII, IX
Thrombin
Prothrombin
Ca2+
Ca2+
Fibrin
Tissue
Factor III
Extrinsic
Pathway
Tissue
Damage
Fibrinogen
Activated Proenzymes,
usually Factor XIII
Platelet
Factor PF-3
Intrinsic
Pathway
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
Prevention with Pluronic F108
PEO
F108 approximate
maximum length: 50nm
PEO
®
HYDROPHILIC
Approximate length of a
red blood cell: 5µm
(500nm)
PPO
HYDROPHOBIC SURFACE
HYDROPHOBIC
How Brush Layer Functions
HYDROPHOBIC
Nisin - Lantibiotic
 Inactivate bacteria by creating a pore and destabilizing the membrane
 Naturally made from bacteria Lactococcus lactis
 Used in food products: preservative, making cheese
 No evidence suggests nisin induces an immunogenic reaction (based on
previous studies)
Hydrophobic Surface
Previous Research
 Change between pluronic coating with nisin before and after
challenged with fibrinogen.
 Two possibilities may account for the lower signal
Purpose
Identify fibrinogen adsorption on non-fouling,
antimicrobial surfaces.
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.
Methodology
 Surface preparation:
Inactivated
Platelets
 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
Fibrinogen
 Parallel flow platelet adhesion tests
Surface
Enzyme Linked Immunosorbant Assay
“Tagged” fibrinogen antibody
detects fibrinogen in a sample,
and then a colorimetric substrate
detects the antibody
Block well with bovine serum
albumin (BSA) or milk.
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.
Results
1.60
Relative Fibrinogen Binding
1.40
1.20
1.00
10-Jul
0.80
18-Aug,BSA
0.60
18-Aug,Milk
0.40
0.20
0.00
S
SN
SF
SFN
Treatments
*Not to scale.
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.
Results
300
fmol FITC / cm 2
250
200
150
100
50
0
S
SN
SF
Treatments
SFN
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.
Results
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.
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).
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.