Bioactivity of Nisin-Loaded Pluronic Microspheres
Challenged by Blood Proteins
Julie Auxier
Dr. Joseph McGuire - Bioengineering
Oregon State University
HHMI Summer 2008
Infected Hospitals
MRSA Infections in 2005
• 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
Community-Associated
Healthcare-Associated
Source: ABCs Population-based surveillance System, Klevens et al. JAMA 2007
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
Common Pathway
Factor X
Factor VII Tissue
Factor Complex
Factor X Activator
Complex
Prothrombinase
Clotting
Factor VII
Clotting Factors
VIII, IX
Thrombin
Prothrombin
Ca2+
Ca2+
Fibrin
Tissue
Factor III
Extrinsic
Pathway
Tissue
Damage
Fibrinogen
Platelet
Factor PF-3
Activated Proenzymes,
usually Factor XIII
Intrinsic
Pathway
Prevention with Pluronic F108
HYDROPHOBIC
SURFACE
PEO
PEO
®
PPO
HYDROPHILIC
HYDROPHOBIC
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
How Brush Layer Functions
HYDROPHOBIC
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
Add nisin
Challenge with blood proteins
Plate samples with diluted
Pediococcus pentosaceus
Quantify with colony counting
Silanized Silica Microspheres
Horse Plasma
γ F108
Nisin
Horse
Plasma
No Nisin
Horse
Plasma
Nisin
Horse
Plasma
M
MN
MF
MFN
MS
MNS
MFS
MFNS
Effect of Nisin on Pediococcus
PED
MFN
MN
MFNS
MNS
Results
Sample Categories Normalized to Pediococcus
Pentosaceus
Colonies of Sample/Colonies of Pediococcus
1.6
1.4
1.2
1
Blank
Untreated Pediococcus
0.8
MFN Treated
MF Treated
0.6
MN Treated
M treated
0.4
0.2
0
0
1
2
3
4
Trial
5
6
7
8
Results
Normalized Pediococcus vs. MFN, MN
Colonies of Sample/Colonies of Pediococcus
1.6
1.4
1.2
1
Untreated Pediococcus
0.8
MFN Treated
MN Treated
0.6
0.4
0.2
0
0
1
2
3
4
Trial
5
6
7
8
Results
Sample Supernatants
Normalized to Pediococcus
Colonies of Sample/Colonies of Pediococcus
1.8
1.6
1.4
1.2
3-Aug-08
1
10-Aug-08
14-Aug-08
0.8
17-Aug-08
21-Aug-08
0.6
24-Aug-08
0.4
28-Aug-08
0.2
0
Untreated
Pediococcus
MFNS Treated
MFS Treated
Sample Category
MNS Treated
MS Treated
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