In the United States over 300 million central venous catheters are used each year 200,000 users of these catheters are subject to lifethreatening complications › Hydrophobic HYDROPHILIC PEO Polyurethane catheters can be coated with polyethylene oxide (PEO) brush layers PEO PPO HYDROPHOBIC association PEO brush layers can resist adsorption of human blood proteins Can also resist adhesion of bacteria HYDROPHOBIC SURFACE Brush layers can be loaded with nisin › Lantibiotic Nisin › Effective against Gram positive bacteria › Does not give rise to resistant strains Contact with blood proteins HYDROPHOBIC 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. 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. Prepare model hydrophobic surfaces by coating (1 micron) silica microspheres with trichlorovinylsilane (TCVS). Coat derivatized surfaces with PEOcontaining triblock polymer chains (BASF Pluronic ®) of varying lengths, and stabilize with gamma irradiation 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) 60 Unchallenged Zeta Potential (– mV) 50 SDS Wash 40 30 20 10 0 TCVS Silanized F108 F68 P105 P65 L101 L61 55 Unchallenged 45 Challenge 1 Zeta Potential (– mV) Challenge 2 35 Challenge 3 25 15 5 -5 -15 TCVS Silanized F108 60 Zeta Potential (– mV) 50 40 Unchallenged Nisin Challenge 1 Challenge 2 Challenge 3 30 20 10 0 -10 F108 - Buffer F108 - Fibrinogen TCVS Silanized Buffer TCVS Silanized - Fibrinogen 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) 25 Carbon Atomic Percent Atomic Percentage 20 Nitrogen Atomic Percent 15 10 5 0 TCVS Silanized F108 F108 w/nisin TCVS Silanized F108 coated F108 coated w/nisin 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 60 Carbon Atomic Percentage Atomic Percentage 50 Nitrogen Atomic Percentage 40 30 20 10 0 TCVS TCVS w/ nisin F108 F108 w/ nisin 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 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 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