Developing a membrane leakage assay for the binding of bacterial lipopolysaccharide by the cationic amphiphilic peptide WLBU2 Public

http://ir.library.oregonstate.edu/concern/honors_college_theses/z316q367q

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  • Sepsis, caused by bacterial release of endotoxin, is a serious blood infection that causes uncontrolled systemic immune response. In North America alone, sepsis kills between 220,000 and 380,000 people each year, more than prostate cancer, breast cancer, and HIV/AIDS combined. Sepsis treatment is complicated and often requires prolonged hospital stays, costing $17 billion annually. There is a need for an external hemoperfusion to device bind to endotoxin molecules, thereby removing them efficiently from the bloodstream without having to add additional cleaning agents to blood. Cationic amphiphilic peptides (CAPs) tethered to non-fouling surfaces in hemoperfusion microchannels hold promise for effective endotoxin removal. WLBU2, an engineered amphipathic CAP, is expected to bind endotoxin in a way that leaves endotoxin vesicles largely intact. This project aimed to develop a membrane-leakage assay to quantitatively measure endotoxin membrane permeabilization in the presence of CAPs. Calcein, self-quenched at 60 mM, was loaded vesicles of lipopolysaccharides (LPS), an analogue for endotoxin. LPS vesicles containing calcein were isolated from unencapsulated dye via dialysis, and stored at 4oC. Successful isolation was confirmed by a measured increase in fluorescence upon the addition of Triton X-100. Percent leakage was calculated by comparing leakage from WLBU2 addition to leakage from Triton X-100 addition (full permeabilization). Dye-loaded liposomes provide great potential for future investigation with other engineered peptides and surface-binding experiments.
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