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Targeting of Oleosin Micelles
Andrew E. FrederickAugust 8, 2013
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Different Drug Delivery VehiclesStructures formed by amphiphilic molecules Nanoparticles
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Why Recombinant?
Complete control over monomer chemistry
Functionalization
Cellular Peptides
RGD
Specific Targeting
Leading to….
Monodisperse
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Objective• To make targeted
micelles from recombinant oleosin that specifically bind to cells– How to target cells?
Ligands– Each mutation is a
polypeptide chain that has a specific function• RGD – binds to integrins• Tat peptide- nitrogen rich
sequences that cause internalization
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Micelles from Oleosin• A form of oleosin in
which the center core has been truncated to 30 amino acids (-30-) have been shown to make micelles (eg. 43-30-63).
• Can this protein be modified for enhanced internalization and cell targeting?
43-30-63
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Expression and Purification
1 2 3 4 5 6 7 8 9
Protein Gel
TAT RGD DRG
Total Protein (1,4,7)
Soluble (2,5,8)
Insoluble, (3,6,9)
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Labeling: Nile Red in Chloroform• The micelles need to be filled
with dyed in order to be seen under microscope
• The Nile Red is hydrophobic
• Intensity readings should be much higher because 10µM is above CMC of 4µM
570 590 610 630 650 670 690 710 7300
5000
10000
15000
20000
25000
PBS (5µL NR)5 µL (10µM)10 µL (10µM25 µL (10µM)50 µL (10µM)
Wavelength (nM)
Inte
nsity
570 590 610 630 650 670 690 710 7300
5000
10000
15000
20000
25000
PBS (5µL NR)5 µL ( 1µM)10 µL (1µM)25 µL (1µM)50 µL (1µM)
Wavelength (nM)
Inte
nsity
Fill interior with dye
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Labeling: Nile Red in Ethanol• Next, the solvent was
exchanged to Ethanol which is miscible in water
• The results were much more promising
• Still no bright enough to see on confocal
560 580 600 620 640 660 6800
50000
100000
150000
200000
250000
1 µL (20µM)5µL (20µM)10µL (20µM)20µL (20µM)
Wavelength (nM)
Inte
nsity
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Labeling: Alexa Fluor 488
• Binds to protein instead of protein encapsulating it
• Used a different protein that was on hand for a proof of concept experiment
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Schematic of Experiment
Incubation Time: 2 hours 4 hours
(-) Control • Cells and Media
(+) Control• Cells and 10µM
polymersomes• Cells and 5µM
polymersomes
• Cells and 10µM polymersomes
Conditional• Cells and 35.5µM
protein• Cells and 35.5µM
protein
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Polymersomes
2 hours
4 hours
5µM 10µM
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Polymersomes
10µM (4 hours)
5µM (4 hours)
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Micelles35µM
2 hours
4 hours
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Micelles35.5 µM for 2 hours
35.5 µM for 4 hours
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New ExperimentIncubation Time:
2 hours 4 hours
• 100%... -30- protein • 100% -30- protein
• 95%..... -30- protein 5%...... RGD
• 95%..... -30- protein 5%...... RGD
• 95%..... -30- protein 5%...... TAT
• 95%..... -30- protein 5%...... TAT
• 90%..... -30- protein 5%...... RGD 5%...... TAT
• 90%..... -30- protein 5%...... RGD 5%...... TAT
* Total protein concentration at 15µM
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Fluorescence images2
hour
s:4
hour
s:
No Chains RGD TAT RGD/TAT
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30 vs. RGD (2 hours)
-30-
RGD
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30 vs. TAT (2 hours)
-30-
TAT
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30 vs. RGD/TAT (2 hours)
-30-
RGD/TAT
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30 vs. RGD/TAT (4 hours)
-30-
RGD/TAT
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Conclusion
• Nile Red loading did not work (have to directly dye)
• Macrophages engulf the micelles without any chains given an appropriate amount of time
• RGD speeds up the intake of micelles
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Next Steps
• Study the kinetics in depth
• Use a cell type that doesn’t mean “big eater” to determine the actual effectiveness of the chains (if RGD binds to cell and if TAT induces internalization)
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Acknowledgement
• Special thanks to Dr. Hammer, Kevin Vargo, Nimil Sood, and the gang