Bioactivity of Nisin-Loaded Pluronic Microspheres Challenged by Blood Proteins Julie Auxier Dr. Joseph McGuire - Bioengineering Oregon State University HHMI Summer 2008

Infected Hospitals Infections are the 4th largest killer in US In the US in 2005 there were: ▫94,360 invasive MRSA infections ▫18,650 associated deaths Costs millions annually Healthcare-Associated Community-Associated Source: ABCs Population-based surveillance System, Klevens et al. JAMA 2007 MRSA Infections in 2005

Infected Hospitals Problems from implanted devices: ▫Clotting ▫Bacterial adhesion ▫Proliferation Treat with heparin and other anticoagulants ▫Risk of platelet depletion, excessive bleeding

Blood Proteins Albumin, globulin, fibrinogen Fibrinogen key in coagulation Intrinsic Pathway Extrinsic Pathway Tissue Damage Tissue Factor III Factor VII Tissue Factor Complex Clotting Factor VII Activated Proenzymes, usually Factor XIII Platelet Factor PF-3 Clotting Factors VIII, IX Factor X Activator Complex Ca 2+ Fibrinogen Fibrin Prothrombin Thrombin Factor X Prothrombinase Common Pathway

Prevention with Pluronic ® F108 HYDROPHOBIC HYDROPHILIC HYDROPHOBIC SURFACE PEO PPO

Irradiating F108 Surfaces Hydrophobic association not permanent Irradiation creates a covalent bond between the F108 and the silanized silica Fluid flow will not wash away F108

HYDROPHOBIC BACTERIA PROTEIN How Brush Layer Functions

Nisin - Lantibiotic Bacteriocin: peptide produced by bacteria to inactivate other like bacteria Naturally made from bacteria Lactococcus lactis Used in food products: p reservative, making cheese

How Nisin Kills Bacteria

Purpose Challenge Nisin-loaded F108 silica microspheres with blood proteins to quantify protein adsorption to the brush layer.

Predictions Microspheres treated with irradiated F-108 will deflect protein adsorption throughout the entire trial; however, the efficacy of nisin treated samples will decline with time.

Efficacy Test: Silanize bare silica microspheres (~1μm in diameter) Covalently bond F108 by γ irradiation Plate samples with diluted Pediococcus pentosaceus Add nisin Challenge with blood proteins Quantify with colony counting

Silanized Silica Microspheres Horse PlasmaNisin γ F108 Nisin Horse Plasma No Nisin Horse Plasma M MNMFMFN MSMNS MFSMFNS

Effect of Nisin on Pediococcus PED MN MFN MFNS MNS

Results

Future Research Further work on behavior of protein adsorption Test platelet adhesion by challenging the same surfaces with platelet rich plasma Apply the coating to devices in vivo

Acknowledgements Dr. Joseph McGuire Matthew Ryder McGuire Lab Group Dr. Skip Rochefort Dr. Kevin Ahern Howard Hughes Medical Institute Johnson Internship Program