Chain Length Effects on Nisin Adsorption to Polyethylene Oxide Layers

1. Chain Length Effects on Nisin Adsorption to Polyethylene Oxide Layers Mitchell Truong Dr. Joseph McGuire, Bioengineering Oregon State University HHMI 2010

2. Problem • In the United States over 300 million central venous catheters are used each year • 200,000 users of these catheters are subject to life-threatening complications

3. Solution HYDROPHILIC • Polyurethane catheters can be coated with polyethylene oxide (PEO) brush layers • Hydrophobic association • PEO brush layers can resist adsorption of human blood proteins • Can also resist adhesion of bacteria HYDROPHOBIC PEO PEO HYDROPHOBIC SURFACE PPO

4. Solution • Brush layers can be loaded with nisin • Lantibiotic • Effective against Gram positive bacteria • Does not give rise to resistant strains

5. Solution BACTERIA PROTEIN HYDROPHOBIC

6. Purpose • To see if PEO brush layers will retain steric repulsive character when loaded with nisin. • To better understand nisin adsorption and elution behavior based on PEO chain length.

7. Hypothesis • If PEO chains are in the brush configuration, a nisin-loaded PEO layer will retain its steric-repulsive character. • If the outermost nisin molecules are eluted from a nisin-loaded PEO layer, the PEO segments extending beyond the level of entrapped nisin will regain their ability to provide a steric repulsive barrier to blood protein adsorption.

8. Methods • Prepare model hydrophobic surfaces by coating (1 micron) silica microspheres with trichlorovinylsilane (TCVS). • Coat derivatized surfaces with PEO-containing triblock polymer chains (BASF Pluronic ®) of varying lengths, and stabilize with gamma irradiation

9. Zeta Potential Analysis • Experimental Design • Contacted with sodium lauryl sulfate (SDS) • Contacted with fibrinogen only • Contacted with nisin and subsequently with fibrinogen • Detect surface changes using zeta potential • Bare silanized surface has a strongly negative charge • Polymer coated surface has a more neutral charge • Nisin has a positive charge • Fibrinogen has a negative charge (lesser)

10. Results, SDS Challenge

11. Results, Fibrinogen Challenge

12. Results, Nisin and Fibrinogen Challenge

13. Energy dispersive spectroscopy (EDS) • Experimental design • Contacted F108 coated spheres with nisin • Can detect surface composition • Uses electron beam to excite electrons of atoms in sample • Energy released indicates presence of specific atoms • Can take images of sample, scanning electron microscope (SEM)

14. Results, EDS

15. Results, SEM TCVS Silanized F108 coated F108 coated w/nisin

16. X-ray photoelectron spectroscopy (XPS) • Experimental design • Contacted with nisin • Can detect surface composition • Uses x-ray beam to excite electrons of atoms in sample • Kinetic energy of ejected electrons is indicative of specific elements • More sensitive than EDS • Can differentiate between same atoms with different bond types

17. Results, XPS

18. Conclusion • Zeta potential analysis showed: • F108 Pluronic attached in a stable fashion to microspheres • The F108 was in a steric repulsive brush configuration • EDS and SEM analyses showed: • F108 presence on surface of Pluronic coated microspheres • Could not detect nisin adsorption in brush layers • XPS showed: • F108 presence on surface • Nisin adsorption detected

19. Future Research • Continue chain length study using other surface analytical approaches • Study the potency, over time, of nisin against bacteria when loaded in F108 brush layers • Study molecular factors that affect peptide integration into PEO brush layers

20. Acknowledgements Special thanks to: • Dr. Joseph McGuire • Dr. Karl “Rat” Schilke • Ben Steyer • Dr. Woo Kul Lee • Brynn Livesay • Julie Auxier • Steve Golledge (Camcor) • Dr. Kevin Ahern • Dr. Skip Rochefort • Howard Hughes Medical Institute • Johnson Scholars