1. Cys-WLBU2; lysed cells Lysed Bacteria Addition PBS Rinse PEO-tethered WLBU2; lysed cells Lysed Bacteria Addition PBS Rinse PEO-tethered WLBU2; intact cells PBS rinse Bacteria Addition Cys-WLBU2; intact cells Bacteria Addition PBS rinse PEO-Tethered Peptides for Capture of Circulating Bacteria and Endotoxin in Sepsis Surface Modification Ramya Raman, Karl F. Schilke Oregon State University, School of Chemical, Biological, and Environmental Engineering SEM images of the treated gold wafers show bacteria-surface interactions. Pseudomonas aeruginosa PA14 was incubated with wafers and rinsed with PBS before fixing, and dried through a series of increased dilutions of ethanol. The samples were then sputtered with Au before imaging. WLBU2 and tethered WLBU2 do show bacteria interaction, however tethered WLBU2 have fewer aggregates, suggesting binding rather than charge attractions. Atomic Force Microscopy (AFM) phase (left) and height (right) images of gold modified using Scheme 2. The height and RMS values are similar, but the phase angle decreases upon addition of PEO, indicating increased interactions with the tip. Phase angles increase again upon conjugation of charged peptides. Scheme 2: Revised tethering scheme for peptides on gold. The use of carboxylic acid-terminated PEO linker eliminates the glutaric anhydride reaction from Scheme 1. Other control peptides (polyarginine and polylysine) were immobilized by the same scheme; Cys-WLBU2 was directly immobilized on gold. Objectives of this study are to compare binding of bacteria by tethered and surface- immobilized WLBU2, and evaluate differences in binding of lysed and whole bacteria. WLBU2 is a synthetic antimicrobial peptide which has antimicrobial activity against a wide spectrum of endotoxins without damage to mammalian cells. The goal of this research is to demonstrate that WLBU2 may be an effective peptide for hemoperfusion treatment of sepsis. Scheme 1: Immobilization of peptides on gold surfaces through a pendant heterobifunctional PEO tether and conventional bioconjugation chemistry. This scheme was used for XPS analysis. CONTROL X-ray Photoelectron Spectroscopy (XPS) of modified gold surface following scheme 1. There is an increase in N 1s peak for WLBU2, C 1s C-O-C peaks for PEO, and an increase in C 1s and N 1s peaks for PEO-WLBU2. Conclusions PEO and WLBU2/Cys-WLBU2 were uniformly spread and immobilized at the surface using schemes 1 and 2. Immobilized WLBU2 binds to Pseudomonas aeruginosa in buffer, suggesting effective capture. SEM and QCM-D data indicate that positively-charged peptides attract bacteria by electrostatic interactions. Whole and lysed cells are bound by both Cys-WLBU2 and PEO-tethered WLBU2. WLBU2 capture of lysed bacteria is substantially greater and faster than binding of whole bacteria cells. Differences in binding kinetics in QCM-D may be due to multiple interactions with tethered peptides, and research is underway to determine the cause and impact of these trends. Further studies will continue to explore bacterial capture, lysis activity and hemocompatibility of WLBU2. Quartz Crystal Microbalance with Dissipation (QCMD) shows the change in 7 th overtone frequency (blue) and dissipation (orange) for binding of lysed (top) and intact (bottom) P. aeruginosa. The surfaces of the sensors were modified ex situ with Cys-WLBU2 and PEO-tethered WLBU2. Changes in mass quickly increased with addition of the lysed bacteria, while capture of whole bacteria was slower. The decrease in dissipation (“softness”) in Panel 1 may be due to multi-point attachment of PEO-WLBU2 to cell fragments. Medical Research Foundation, National Science Foundation PFI-AIR program Morgan Brown, Tal Schraf, Minot Lab for AFM support Stephen Golledge and Joe Baio Lab group for XPS support Teresa Sawyer, Peter Eschabach, EM Lab Schilke Lab group Au Av:65.4° RMS: 65.3° Au (control) Av:30.1° RMS: 30.2° PEO Av:68.0° RMS: 68.0° Cys-WLBU2 PEO-WLBU2 Av:65.0° RMS: 65.0° PEO-Polylysine Av:79.0° RMS: 80.1° PEO-Polyarginine 1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide HCl (EDC) N-Hydroxysulfosuccinimide (Sulfo-NHS ) Au WLBU2 Au Amine terminated PEO (Polyethylene Oxide) Glutaric Anhydride Au surface RMS: 1.316nm PEO surface RMS: 1.595nm PEO-WLBU2 RMS: 3.129nm Cys-WLBU2 RMS: 1.83nm PEO-Polyarginine RMS: 2.869nm PEO-Lysine RMS: 4.636nm Au Carboxylic Acid terminated PEO (Polyethylene Oxide) Gold surface Au WLBU2 1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide HCl (EDC) N-Hydroxysulfosuccinimide (Sulfo-NHS ) Acknowledgements BackgroundSurface AnalysisPreliminary Results Au control Cys-WLBU2 modified PEO-NH 2 modified PEO-WLBU2 modified C 1s N 1s C 1s N 1s C 1s N 1s C 1s N 1s

3. WLBU2 Lysed Bacteria Addition PBS Rinse PEO tethered WLBU2 Lysed Bacteria Addition PBS Rinse PEO tethered WLBU2 PBS rinse Bacteria Addition WLBU2 Bacteria Addition PBS rinse

9. WLBU2 Bound to the SurfaceAddition of lyed PseudomonasPBS Wash

10. jgi

11. Au NHS (N-HydroxySulfoSuccinimide) 1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide HCl Au Carboxylic Acid terminated PEO (Polyethylene Oxide) Gold surface Au WLBU2 Au NHS (N-HydroxySulfoSuccinimide) 1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide HCl Au Carboxylic Acid terminated PEO (Polyethylene Oxide) Gold surface Au WLBU2 Gold surface 1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide HCl NHS (N-HydroxySulfoSuccinimide) Au WLBU2 Au Amine terminated PEO (Polyethylene Oxide) Glutaric Anhydride