1. Provided by Central Web Services 541-737-1189 http://oregonstate.edu/cwshttp://oregonstate.edu/cws 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 BACKGROUND AND INTRODUCTION METHODS SUPPLEMENTAL METHODS AND RESULTS CONCLUSIONS AND FUTURE WORK RESULTS AND DISCUSSION Many thanks to: Biomaterials/Biointerfaces Group: Dr. Joe McGuire, Dr. Karl “Rat” Schilke, Dr. Michelle Bothwell, Julie Auxier, Matt Ryder BASF for providing the Pluronics® Allvivo Vascular for research partnership Dylan Stankus Dr. Philip Harding ACKNOWLEDGEMENTS 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. ANTIBIOTIC INTEGRATION AND ELUTION PROTEIN REPULSION CHALLENGE 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. 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. I mplantable medical devices such as central venous catheters are susceptible to bacterial adhesion and protein fouling, which cause device failure. 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. Protein Fouling: Blood proteins (such as fibrinogen) adhere to catheter surface and initiate the coagulation cascade, resulting in thrombosis. Central Venous Catheter Solution: 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. HYDROPHOBIC HYDROPHILIC PEO PPO HYDROPHOBIC SURFACE Modified Surface with Pluronic® Brush Project Objectives: 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. Surface Preparation 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 1 micron 141 44 Pluronic® F108 80 27 Pluronic® F68 Sample Preparation 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. Short: (27-30 PO monomers) Pluronic® surfactant EO monomers per PEO chain PO monomers per PPO chain L61330 P651930 F688027 Pluronic® surfactant EO monomers per PEO chain PO monomers per PPO chain L101756 P1053856 F10814144 Long: (44-56 PO monomers) PEO PPO Surface Preparation Sample Preparation and Zeta Potential Analysis = Zeta Potential Measurement TCVS Silanization, coating w/Pluronic®, and irradiation Fibrinogen Challenge 3 Fibrinogen Challenge 2 Fibrinogen Challenge 3 Fibrinogen Challenge 2 Fibrinogen Challenge 1 Buffer Challenge 3 Incubate w/ Nisin Buffer Challenge 2 Buffer Challenge 1 Fibrinogen Challenge 1 SDS Wash Summary of results: 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. Enzyme-Linked Immunosorbent Assay (ELISA) 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. Figure 4: shows results of ELISA experiments performed with uncoated and F108- coated 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. + + + + Positive + + + + - - - - - Negative - - - - - Laser Zeta Potential Analysis 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. Left: ZetaPALS analyzer. Right: Diagram of particle movement and detection during zeta potential analysis www.microscopesblog.com/2009/08/elisa.html http://uwmedicine.washington.edu/Patient-Care/Our-Services/ Nisin, the antibacterial peptide loaded in the Pluronic® brush http://en.wikipedia.org/wiki/File:Nisin_1WCO.png Future Work: 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. ELISA Antibody Tagging STABILITY CHALLENGE