Functionalization of Surfaces with Nisin
in a Poly[ethylene oxide] brush layer
Matt Ryder
Dr. Joe McGuire – BioEngineering
OSU
HHMI Summer ‘07
Hydrophobic Surface
Background – Instances of Infection1


Infection in hospitals is the fourth largest
killer in the US
2 million patients contract infections in
hospitals each year…about 103,000 die as
a result


228 infections/hour…12 deaths
Cost? $30.5 billion each year
Background

Three issues with implanted devices
Clot Formation
 Bacterial Adhesion
 Cell Proliferation


Currently, methods
to counteract
include:

Loading patients
with heparin or
antibiotics.
Background

These issues are directly related,
both are initiated by adsorption
events.
Background – Brush Layer

F108 creates brush layer that protects
against protein adsorption.
 Protein adsorption can result in
very different outcomes, from
benign surface coatings, to large
clot formation (therefore risk of
stroke).
Hydrophobic Surface
Brush Layer – Protein Repellant
Bare Surface
F108 Coated Surface
Bare Surface
F108 Coated Surface
A.
B.
Background – Nisin
Nisin is a small antimicrobial peptide produced by
strains of Lactococcus lactis subsp. lactis. Nisin kills
Gram positive bacteria through a multistep process
that destabilizes the phospholipid bilayer of the cell
and creates transient pores. The efflux of low
molecular weight compounds from the cytoplasm
and subsequent dissipation of membrane potential
rapidly kills the targeted bacterium.
Nisin - Mechanism
Background – Nisin & Brush Layer

Goes against function of brush layer
Hydrophobic Surface
Hydrophobic Surface
Background – Product Activity
Hydrophobic Surface
Hypothesis

If a surface can be chemically or
functionally modified to adsorb and
retain the Lantibiotic Nisin,
antimicrobial activity and anti
clotting function will be higher as
compared to current coating
methods.
Procedure
microspheres
F108
Nisin
2 days
x # of days
Results





Optimized concentrations of F108, Nisin
and microspheres in solution.
Conducted serial dilution tests to find
optimum concentration of Pediococcus.
Researched literature for background
knowledge and future effectiveness
Standardized procedures to decrease
variability.
Obtained valuable insight on Nisin activity
from longevity testing.
Results
10-6 dilution w/o Nisin
10-4 dilution w/ Nisin
Dilution optimized for
30-300 colonies
Results
14
Colony Forming Units
12
F108-coated microspheres+nisin
Uncoated microsphere+nisin
10
8
6
4
2
0
1 day
4 days
Incubation time
Results
Colony Forming Units
200
Nisin+F108 (no microspheres)
Nisin only (no microspheres)
150
100
50
0
1 day
4 days
Incubation time
Future Research




28 day trials with current procedure
Blood serum studies to test
longevity of Nisin in physiological
conditions
Tests with EGAP rather than F108, a
more clinically accepted polymer
in vitro studies using catheters
Acknowledgements

Special Thanks to:
Dr. Joe McGuire – Mentor
 Dr. Christine Kelly
 Karle Schilke
 Dr. Jeff Tai – Protocols and instruction
 Dr. Kevin Ahern – HHMI Program
 The Howard Hughes Medical Institute

References
1. Committee to Reduce Infectious
Diseases
http://www.hospitalinfection.org/essentialfacts.shtml
Pictures
1. http://www.flickr.com/photos/gaspirtz/384254225/
2. www.altham.com/html/food_hygiene_cartoons.html
3. http://www.sciencestuff.com/prod/L-p-Empty/1001-20
4. http://www.flickr.com/photos/rdbkorn/85401201/
5. http://www.bergoiata.org/fe/favs/Bacteria.jpg
6. www.sigmaaldrich.com/img/assets/4261/micro_7.gif