The overall goal of this procedure is to design a delivery system that can deliver small molecule drugs like siRNA to the brain. This method can answer key questions in the neurodegeneration field such as Is siRNA a viable therapeutic option for protein misfolding diseases? and Can we design a reliable drug delivery system to the brain?
The main advantage of creating a liposomal drug delivery system is that we can deliver our siRNA intravenously or through the blood stream rather than directly through neuronal tissue which results in neuronal damage. The implications of this technique extends towards therapeutics and diagnostics of neurodegenerative diseases because this technique allows for specific delivery across the blood-brain barrier, specifically to acetylcholine-expressing neurons. Visual demonstration of this technique is critical as mouse tail vein injections are difficult to learn due to the small size of the mouse tail vein.
Begin by preparing a one-to-one DOTAP to cholesterol ratio. For a foreign animal liposome preparation, mix two nanomoles of DOTAP and two nanomoles of cholesterol into ten milliliters of a one-to-one chloroform to methanol solution. Then, evaporate nine milliliters of the chloroform and methanol solution using nitrogen gas.
Evaporate the last one milliliter of solvent in a fume hood overnight without gas. The next day, a thin lipid film should be visible on the bottom of the flask. Heat both this flask with the lipid film and ten milliliters of a ten percent sucrose solution to 55 degrees Celsius.
Then, slowly pipette the heated sucrose solution onto the thin lipid film while gently swirling the flask. Next, assemble an extruder with one micron filters according to the manufacturer's instructions. Then, add one milliliter of the heated liposome suspension to one of the syringes on the extruder.
Then pass the suspension between the two syringes 11 times. While keeping the liposome suspension heated for easier sizing, size the liposomes through 45 micron and a 2 micron filter To generate large, unilamellar vesicles, next add 20 microliters of the four nanomole liposome suspension to 200 microliters of a 20 micromolar stock siRNA solution and incubate for ten minutes at room temperature. Then add 80 microliters of a 500 micromolar stock of RVG-9R targeting peptide to the siRNA liposome solution and incubate for ten minutes at room temperature.
Prepare a foreign animal lipsomoe suspension by mixing 2.2 nanomoles of either DSPE or DOTAP into ten milliliters of a two-to-one chloroform to methanol solution with 1.6 nanomoles of cholesterol and 2 nanomoles of DSPE peg. Afterwards, apply nitrogen gas and when most of the solvent has been evaporated, leave the flask in the fume hood overnight. Then, slowly add ten milliliters of 1X PBS heated to 75 degrees Celsius to the thin lipid film.
Allow the lipids to rehydrate at 75 degrees Celsius for at least one hour before sizing. After sizing the liposome suspension using an extruder as before, pipette 20 microliters of each of the four nanomole liposome suspensions into single use aliquots and lyophilize for 30 minutes using a bench top freeze dryer. Next, add 1.4 microliters of a 26.6 micromolar solution of protamine sulfate to 200 microliters of 20 micromolar stock siRNA solution and incubate for ten minutes at room temperature to condense the siRNA.
Following the incubation, rehydrate DSPE palets with 201.4 microliters of siRNA protamine solution. Rehydrate the DOTAP palets with 200 microliters of a foreign animal stock solution of siRNA. Incubate each liposome siRNA solution for ten minutes at room temperature.
Next, add ten microliters of a 60 millimolar EDC solution to each tube of rehydrated liposomes. Then add ten microliters of 150 millimolar sulfo-NHS solution to the liposome siRNA solution for both DOTAP and DSPE palets. Incubate for two hours at room temperature.
Lastly, add 80 microliters of 500 micromolar stock of RVG-9R peptide to the siRNA liposome cross linking solution and incubate for ten minutes at room temperature. After anesthetizing a mouse using two to three percent isoflurane in oxygen cover the eyes of the mouse with apthalmic ointment and confirm an anesthetization by performing a toe pinch. Proceed only if a reaction is absent.
While maintain anesthesia, place the mouse on its back or side to access the tail vein. Wipe the tail with 70 percent ethanol and then use a 26 gauge insulin syringe to inject 300 microliters of LSPCs or palets into the tail vein. Begin injecting distally and move proximally if the vein collapses or ruptures.
Place the mouse in a clean cage until it maintains sternal recumbency. Monitor the animal for several hours after injection to ensure anesthesia wears off and there are no ill effects of the treatment. After allowing the LSPCs or palets to circulate for at least 24 hours and then euthanizing the mouse, dissect out half a hemisphere of the brain.
Then place the half hemisphere and 2.5 milliliters of fax buffer onto a 40 micron mesh cell strainer and use the plunger of a syringe to press the tissue through the filter into a petri dish. Rinse the cell strainer and petri dish with an additional 2.5 milliliters of fax buffer. Then transfer the single cell suspension to a 15 milliliter conical tube and place on ice.
Next, pipette 100 microliters of the cell suspension into micro centrifuge tube and centrifuge for five minutes at 350 times G.After discarding the supernatant, resuspend the cell pellet in one milliliter of fax buffer and centrifuge again. Then, add another milliliter of fax buffer and place the cell suspension on ice. Now, incubate the cells in 100 microliters of rat anti-mouse Fc block in fax buffer for 30 to 60 minutes on ice.
After pelleting and washing as before, add 100 microliters of fluorescent primary anti-body solution and place on ice for 30 to 60 minutes. After a final wash, resuspend the cells in one mililiter of RVC lysis buffer for one minute. Then, pellet the cells.
After discarding the buffer, resuspend in one milliliter of fax buffer and place on ice. The cells are now ready for fax analysis. Mice treated with siRNA delivered via LSPCs showed a significant decrease of cellular prion protein levels compared to a PBS control which shows while type cellular prion protein levels.
The LSPCs treated mice showed cellular prion protein levels close to that of a prion protein knock out mouse. Across three experiments, mice treated with LSPCs showed significant decreases in cellular prion protein levels as compared to to PBS while type controls. While attempting this procedure, it's important to remember that the liposomes can be stored in the fridge for several months, but the complete LSPCs must be used within 24 hours after assembly.
After its development, the generation of LSPCs or palets will pave the way for researchers to explore more therapeutic options in the field of neurodegenerative diseases.
