Chain Length Effects on Nisin Adsorption to Polyethylene Oxide Layers

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Chain Length Effects on Nisin Adsorption to
Polyethylene Oxide Layers

• Mitchell Truong
• Dr. Joseph McGuire, Bioengineering
• Oregon State University
• HHMI 2010

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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

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Solution

• 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 SURFACE
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Solution

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

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Solution
HYDROPHOBIC
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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.

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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.

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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

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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)

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Results, SDS Challenge
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Results, Fibrinogen Challenge
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Results, Nisin and Fibrinogen Challenge
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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)

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Results, EDS
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Results, SEM
F108 coated
TCVS Silanized
F108 coated w/nisin
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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

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Results, XPS
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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

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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

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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