Transcript Slide 1

Implantable Medical Device Coatings for Prevention of Infection and Thrombosis
Aleksandr Gerasimenko, Andrew Sinclair, and Benjamin Steyer
School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97333
Mentor: Dr. Joseph McGuire
METHODS
BACKGROUND AND INTRODUCTION
Pluronic® F108
Surface Preparation
Implantable medical devices such as central venous
catheters are susceptible to bacterial adhesion and protein
fouling, which cause device failure.
• Clean silica microspheres by acid/base wash.
• Silanize using trichlovinylsilane (TCVS) to add vinyl groups
• Incubate microspheres with with Pluronic® to coat.
• Gamma irradiate samples (0.3 Mrad) to covalently attach
Pluronics® to surface
• Bacterial Adhesion: biofilm formation and local infection
on the catheter. It has been estimated that 200,000
infections occur annually from central venous catheter use
alone, causing up to 20,000 deaths per year in the ICU, at a
cost that may exceed $1 billion.
PEO
141
44
Pluronic® F68
80
27
• Assay used to determine relative quantity of fibrinogen alone attached to surface after incubation
(challenge).
• Pluronic® attachment to silicon wafer surfaces by incubation and irradiation.
• Color linked anti-fibrinogen antibody incubated with sample post fibrinogen challenge.
• The absorbance of each sample (490 nm) was used to calculate the adsorbed amount of fibrinogen.
1.2
Sample Preparation
HYDROPHILIC
+ + + + Positive + + + +
Zeta Potential Analysis
Modified Surface with Pluronic® Brush
0.8
0.6
0.4
0.2
0.0
Silica
• Zeta potential measured using Zeta
PALS analyzer (Brookhaven)
• Particle charge, which is dependant
on identity or quantity of material
adsorbed to the surface, is
determined by velocity of particles
as they move towards an electrode.
HYDROPHOBIC
Normalized ELISA Signal
Central Venous Catheter
PPO
1.0
• Microspheres incubated in solutions with
known concentrations of nisin or human
fibrinogen for 1 hr.
• Washes performed by subsequent
centrifugation and resuspension in clear
buffer prior to analysis.
http://uwmedicine.washington.edu/Patient-Care/Our-Services/
PEO
Enzyme-Linked Immunosorbent Assay (ELISA)
1 micron
• Protein Fouling: Blood proteins (such as fibrinogen) adhere
to catheter surface and initiate the coagulation cascade,
resulting in thrombosis.
HYDROPHOBIC SURFACE
SUPPLEMENTAL METHODS AND RESULTS
Silica + Nisin
Silica + F108
Silica + F108 +Nisin
Figure 4: shows results of ELISA experiments performed with uncoated and F108coated silica samples, in the presence and absence of adsorbed nisin. These results
suggest that the presence of nisin in the PEO layer evoked a fibrinogen loading that
is not significantly greater than with PEO alone.
Laser
ELISA Antibody Tagging
www.microscopesblog.com/2009/08/elisa.html
- - - - - Negative - - - - -
CONCLUSIONS AND FUTURE WORK
Left: ZetaPALS analyzer. Right: Diagram of particle movement and detection during zeta
potential analysis
Solution:
25
Nisin, the antibacterial peptide
loaded in the Pluronic® brush
40
15
10
5
Surface
Preparation
Challenge 1
30
Challenge 2
25
Challenge 3
20
10
Pluronic®
surfactant
5
L61
P65
F68
0
F68
P105
P65
L101
Figure 1: Zeta potential results for microspheres coated with Pluronics® F108, F68,
P105, and P65. Zeta potentials were taken pre- and post-SDS challenges to determine
the quality/integrity of coatings. Results indicate Pluronics® are stably and covalently
attached to microsphere surfaces.
F108
Fibrinogen
Challenge 1
Fibrinogen
Challenge 2
Fibrinogen
Challenge 3
Fibrinogen
Challenge 1
Fibrinogen
Challenge 2
Buffer
Challenge 1
Buffer
Challenge 2
Sample Preparation
and Zeta Potential
Analysis
Provided by Central Web Services
541-737-1189
http://oregonstate.edu/cws
Fibrinogen
Challenge 3
= Zeta Potential Measurement
Buffer
Challenge 3
3
19
80
30
30
27
Long: (44-56 PO monomers)
Pluronic®
surfactant
L101
P105
F108
EO monomers
per PEO chain
PO monomers
per PPO chain
7
38
141
56
56
44
• Continue characterization of coatings to study the effects of
Pluronic® chain length on fibrinogen adsorption and nisin
adsorption and elution.
• Assess performance of Pluronic® brushes made with L61,
P65, L101, and P105.
• Use additional quantitative methods (XPS and TOF-SIMS) to
determine quantity of fibrinogen and nisin at the brush layer
interface after fibrinogen challenge.
ACKNOWLEDGEMENTS
Challenge 1
20
PO monomers
per PPO chain
Unchallenged
Nisin
Zeta Potential (– mV)
SDS Wash
25
F68
Figure 2: Fibrinogen challenges for TCVS silanized (γ-irradiated), F108, and F68-coated
microspheres. Fibrinogen adsorption is indicated by a positive shift in zeta potential.
TCVS silanized microspheres displayed pronounced fibrinogen adsorption after one
challenge. F108 and F68 zeta potentials did not display a significant positive shift after
each trial, as expected, indicating the presence of an intact brush coating.
ANTIBIOTIC INTEGRATION AND ELUTION
Incubate w/
Nisin
EO monomers
per PEO chain
-5
TCVS Silanized
Challenge 2
Many thanks to:
Challenge 3
15
Biomaterials/Biointerfaces Group:
10
Dr. Joe McGuire, Dr. Karl “Rat” Schilke, Dr. Michelle Bothwell,
Julie Auxier, Matt Ryder
5
BASF for providing the Pluronics®
Allvivo Vascular for research partnership
0
F108 - Buffer
F108 - Fibrinogen
F68 - Buffer
F68 - Fibrinogen
Figure 3: Zeta potential results prior to, and after nisin (antibiotic) integration for F108 and F68-coated microspheres. Nisin
incubation causes a notable shift toward the positive in zeta potential as nisin integrates into the brush coating in multilayer
quantities. Subsequent challenges with buffer or fibrinogen indicate possible elution of nisin from the brush layer.
1
PPO
Future Work:
Short: (27-30 PO monomers)
15
0
F108
TCVS Silanization,
coating w/Pluronic®,
and irradiation
Unchallenged
35
20
Zeta Potential (– mV)
• Verify covalent attachment stability of various Pluronics® to surface with SDS challenge.
• Assess repulsive character of Pluronic® brush layers produced.
• Determine pendant chain length and spacing effects on nisin elution, and fibrinogen interactions
at nisin-loaded brush interface.
PROTEIN REPULSION CHALLENGE
No SDS
SDS washed
http://en.wikipedia.org/wiki/File:Nisin_1WCO.png
Project Objectives:
STABILITY CHALLENGE
• SDS challenges indicated that Pluronics® F108, F68, P105, and P65 were covalently
attached to microsphere surface.
• Fibrinogen challenge confirmed repulsive character of F108 and F68 brush coatings.
• Results indicate nisin elution from F108 and F68 brush layers is independent of
fibrinogen presence in challenge buffer.
• Supplemental data, combined with our results, indicates that nisin loading does
not compromise repulsive character of Pluronic® brush layers.
PEO
RESULTS AND DISCUSSION
Zeta Potential (– mV)
A biomedical surface coating consisting of Pluronic® tri-block
copolymers covalently attached to the catheter surface in a “brush”
configuration to sterically repel proteins and prevent hydrophobic
association. Nisin, an antibacterial peptide, will be loaded in this
brush to prevent infection.
PEO
Summary of results:
Dylan Stankus
Dr. Philip Harding