Bioactivity of Nisin-Loaded Pluronic Microspheres Challenged by Blood Proteins

Julie AuxierDr. Joseph McGuire - BioengineeringOregon State UniversityHHMI 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 annuallyHealthcare-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

Ca2+

Ca2+

FibrinogenFibrin

Prothrombin Thrombin

Factor X

Prothrombinase

Common Pathway

Prevention with Pluronic® F108

HYDROPHOBIC

HYDROPHILIC

HYDROPHOBICSURFACE

PEO

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

PROTEI

N

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: preservative, making cheese

Hydrophobic Surface

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 Plasma Nisinγ F108

NisinHorse Plasma

No Nisin

Horse Plasma

Horse Plasma

M MN MF MFN

MS MNS MFS MFNS

Effect of Nisin on Pediococcus

PED MNMFN

MFNS MNS

Results

Results

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

AcknowledgementsDr. Joseph McGuire

Matthew Ryder

McGuire Lab Group

Dr. Skip Rochefort

Dr. Kevin Ahern

Howard Hughes Medical Institute

Johnson Internship Program