Targeting of Oleosin Micelles

Andrew E. FrederickAugust 8, 2013

Different Drug Delivery VehiclesStructures formed by amphiphilic molecules Nanoparticles

Why Recombinant?

Complete control over monomer chemistry

Functionalization

Cellular Peptides

RGD

Specific Targeting

Leading to….

Monodisperse

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

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

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)

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

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

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

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

Polymersomes

2 hours

4 hours

5µM 10µM

Polymersomes

10µM (4 hours)

5µM (4 hours)

Micelles35µM

2 hours

4 hours

Micelles35.5 µM for 2 hours

35.5 µM for 4 hours

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

Fluorescence images2

hour

s:4

hour

s:

No Chains RGD TAT RGD/TAT

30 vs. RGD (2 hours)

-30-

RGD

30 vs. TAT (2 hours)

-30-

TAT

30 vs. RGD/TAT (2 hours)

-30-

RGD/TAT

30 vs. RGD/TAT (4 hours)

-30-

RGD/TAT

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

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)

Acknowledgement

• Special thanks to Dr. Hammer, Kevin Vargo, Nimil Sood, and the gang