TitleTowards a Selectively Bioactive Surface for the Removal of Circulating Endotoxin in Blood
Publication TypeThesis
Year of Publication2017
AuthorsRaman, R
UniversityOregon State University
CityCorvallis, Or.
Thesis TypeDissertation

Sepsis is an inflammatory reaction occurring throughout the human body to infections caused by bacteria, fungi, and or other forms of pathogens. It is essential to find an alternative treatment method for sepsis, to lessen the dependence on antibiotics. Hemoperfusion is an absorbent device that removes select targets from blood, when passed through it. Recent studies are evaluating methods for treatment of antibiotic resistant bacteria using antimicrobial peptides and bacteriophage (viruses against bacteria) proteins, as an alternate to antibiotic treatment. A promising method is to combine a hemoperfusion device coated with PEO modified antimicrobial peptides and or bacteriophage proteins to remove endotoxins, released by bacteria but prevent plasma protein platelet adsorption through the PEO brush layer effect. For this purpose, antimicrobial peptide WLBU2 and bacteriophage derived protein Abc2 were studied. This study demonstrates the ability of WLBU2 to retain endotoxin and bacteria binding abilities when PEO is tethered to a surface. Circular Dichroism demonstrated that the secondary helical structure was retained in the presence of LPS when the peptide was PEGylated indicating that the flexibility of the peptide is not inhibited when PEGylated. PEGylated gold surfaces with terminal WLBU2 demonstrated bacteria and endotoxin capture through Scanning Electron Microscopy (SEM) and Quartz Crystal Microbalance with Dissipation (QCMD). Abc2 protein was modified using Genetic Code Expansion (GCE) to incorporate unnatural amino acid Azide-Phe, which uses click chemistry to bind to specific functional groups. This prevents non-specific binding of the protein on the surface, compared to standard bioconjugation methodologies that enable multiple conformations of the protein to the surface. Click chemistry retains the structure and function of the protein. Surface analysis methods using X-ray Photoelectron Spectroscopy (XPS) and QCMD demonstrated the ability of the protein to be immobilized using click chemistry to F127 with end group terminal Alkyne. LPS binding capabilities of the Abc2 modified surface was demonstrated using FITC-LPS solution depletion assay and QCMD. Polybutadiene-Polyethylene Oxide (PBD-PEO) co-polymers were studied for hemocompatibility on a C18 silane surface. The PBD groups can covalently bind using irradiation to biomedical plastics, unlike current Pluronics, which require expensive and toxic surface coatings. Three varied sizes of copolymers were studied and compared to F127. AFM and QCMD was used to monitor the immobilization dynamics of PEO-PBD diblocks compared to Pluronic F127. Generally, the diblocks had a much lower surface coverage, but P5431 exhibited similar topology and coverage as F127. Hemocompatability was studied by fibrinogen repulsion experiments using QCMD and FITC-fibrinogen solution depletion assays. Patelet activation was studied by SEM. P5843 was more efficient at fibrinogen repulsion but was not as efficient at platelet repulsion as P5431, due to the PEO length difference. These results provide further analysis of the endotoxin binding ability of tethered WLBU2, as well as offers a method to effectively tether Abc2 protein to a Pluronic surface for LPS binding. Finally, this work exhibits the hemocompatablility of covalently bound PEO-PBD for a hemoperfusion device. Overall, these results provide a methodology towards a commercial bioactive surface for the treatment of sepsis.