Targeted siRNA Delivery to Neurons for Prion Protein Silencing Using Liposomal Complexes

Samenvatting

Source: Bender, H. R., et.al. Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes. J. Vis. Exp. (2016)

This video demonstrates the targeted delivery of prion protein (PrP) siRNA to neurons using liposomal siRNA-peptide complexes (LSPCs). Cationic liposomes electrostatically bind PrP-targeting siRNA, which is further stabilized by the RVG-9r peptide for receptor-mediated transport across the blood-brain barrier. Once inside the brain, LSPCs undergo endocytosis in neurons, releasing siRNA to initiate RNA interference, reducing PrP expression and interrupting prion disease progression.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of LSPCs

1. Use a 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium-propane): cholesterol ratio for LSPCs. For a 4 nmole liposome preparation, mix 2 nmoles of DOTAP and 2 nmoles of cholesterol into 10 ml of a 1:1 chloroform:methanol solution in a flask.
2. Evaporate 9 ml of the chloroform:methanol solution using N₂ gas in a fume hood. Evaporate the last 1 ml of solvent in a fume hood overnight without gas. Observe a thin lipid film on the bottom of the flask.
3. Heat 10 ml of a 10% sucrose solution to 55 °C.
4. Pour the heated 10% sucrose solution onto the thin lipid film slowly, i.e. 1 ml/min, while gently swirling the flask. Maintain the 10% sucrose and flask with lipids at 55 °C so that the lipid molecules remain in the gel-liquid phase. Rehydration results in multilamellar vesicle (MLV) formation.
5. Allow lipids to rehydrate at 55 °C for at least one hr before sizing the liposomes.
6. Assemble an extruder per manufacturer's instructions with 1.0 µm filters or use 1.0 µm syringe filters for sizing.
7. Add 1 ml of the heated liposome suspension to one of the syringes on the extruder and pass the suspension between the two syringes 11 times. Make sure that the suspension is in the opposite syringe than the starting syringe after the 11 passes.
8. Size the liposomes again through 0.45 µm then 0.2 µm filters to generate large unilamellar vesicles (LUVs). Keep the liposome suspension heated for easier sizing.
9. Incubate 20 µl of the 4 nmole liposome suspension (200 µM) with 200 µl of a 20 µM (4 nmole total) stock siRNA solution for 10 min at room temperature.
10. Incubate 80 µl of a 500 µM (40 nmole total) stock RVG-9r solution with the siRNA/liposome solution for 10 min at RT. LSPCs should be used as soon as possible but can be used up to several hours after preparation. Store LSPCs at 4 °C for several hr after preparation.

2. Injecting Mice with LSPCs

1. Expose mice for 5-10 min to a heat lamp to dilate vasculature.
2. Anesthetize the mouse with a 2-3% isofluorane/oxygen flow 5-10 min before injection, and during injection. Confirm anesthetization when the animal is no longer ambulant, and respirations have slowed, by doing a toe pinch. Place the mouse on its back or side to access the tail vein. Use vet ointment on mouse's eyes to prevent drying while under anesthesia.
3. Wipe/spray the tail with 70% EtOH and inject 300 µl of LSPCs onto the tail vein of the mouse using a 26 G insulin syringe. Start injecting distally into the tail vein and move proximally if the vein collapses or ruptures.
4. Place the mouse in a clean cage until it maintains sternal recumbency. Do not place the mouse with other cage mates until it has fully recovered. Mice should recover in 5 to 15 min. They can be placed on a heating pad or under a heat lamp to maintain body temperature if recovery takes longer. Mice should be monitored closely to safeguard against over-heating.
5. Monitor the animal several hours after injection to ensure the anesthesia wears off, and there are no ill effects of the treatment. Allow the LSPCs to circulate for at least 24 hr.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
DOTAP lipid	Avanti Lipids	890890
Cholesterol	Avanti Lipids	700000
Chloroform	Fisher Scientific	AC268320010
Methanol	EMD Millipore	113351
N₂ Gas	AirGas
Sucrose	Fisher Scientific	S5-500
Extruder	Avanti Lipids	610023
1.0, 0.4, and 0.2 μm filters	Avanti Lipids	610010, 610007, 610006