This protocol allows the production of safe and efficient nanoliposomes which can be used as transfection vehicles for therapeutic messenger RNA. These nanoliposomes facilitate mRNA uptake in cells resulting in increased protein expression levels without affecting cell viability. The main advantage of this technique is fast and easy generation of stable nanoliposomes with defined size and homogenous distribution.
Furthermore, they protect the encapsulated mRNA from degradation and lead to prolonged translation. Demonstrating the procedure will be Antonia Link, a PhD student from our laboratory. To begin this procedure, prepare stock solutions for the lipids, DC-cholesterol and DOPE by dissolving each in chloroform to a final concentration of 25 milligrams per milliliter.
Mix 40 microliters of the dissolved DC-cholesterol with 80 microliters of the dissolved DOPE in a glass flask. Vaporize the chloroform under an argon gas flow for 15 minutes. Next, fill a desiccator with silica gel.
Place the open glass flask inside and apply a vacuum overnight to ensure that the remaining chloroform is evaporated. The resulting lipid film will form inside the glass flask. The next day, add one milliliter of nuclease free water to rehydrate the lipid film.
And vortex the suspension for 15 minutes. Place the suspension into a sonication bath for one hour. After this, assemble the mini extruder according to the manufacturers instructions.
Fill a syringe with the liquid suspension and place it one side of the extruder. Place an empty syringe on the other side. Press the lipid suspension through the membrane from one syringe to the other, approximately 20-25 times to extrude the suspension.
Then, store the nanoliposomes in a glass flask at four degrees Celsius until ready to use. First, thaw the synthetic mRNA on ice. Vortex the frozen mRNA and then centrifuge it shortly before opening the tube.
Then, mix one microgram of the synthetic mRNA with various amounts of nanoliposome suspension. Centrifuge briefly and incubate at room temperature for 20 minutes for nanolipoplex formation. Then, add milliliter of regular cell medium to the nanolipoplex's and mix by pipetting up and down.
Prepare the cells one day before transfection. Therefore, plate 150, 000 A549 cells into each well of a 12 well plate one day prior to transfection. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and regular cell medium for 24 hours.
Then next day, wash each well of prepared cells one time with one milliliter of PBS. Add one milliliter of a prepared nanolipoplex mixture to one of the wells containing the A549 cells. Incubate at regular conditions for 24 hours to analyze the transfection efficiency, or for 24-72 hours to analyze the cell viability after transfection.
Remove the supernatant and wash each well with one milliliter of PBS to remove the remaining nanoliposomes. To prepare the cells for cytometry, first add 500 microliters of trypsin EDTA to each well and incubate at 37 degrees Celsius for three minutes to trypsinize the cells. Next, add 500 microliters of regular FBS-containing medium to stop the process and inactivate the trypsin.
Centrifuge the cells at 400 times G for five minutes. And carefully remove the supernatant without disturbing the cell pellet. Wash the cells with one milliliter of PBS.
Then re-suspend the cells in 300 microliters of cell fixation solution and transfer them into flow cytometry tubes. Use a flow cytometer to analyze the cells at 488 nanometers. To prepare the cells for fluorescence microscopy, add one milliliter of chilled methanol to each well to fix the cells.
Add 500 microliters of a 300 nanomolar solution of DAPI dissolved in PBS to each well. Incubate in the dark for five minutes. After this, remove the DAPI solution and wash the cells again with 100%methanol that was previously stored at minus 20 degrees Celsius.
Use a fluorescence microscope to analyze the cells using the excitation and emission wavelengths shown here. In this study, nanoliposomes are generated with high encapsulation efficacy for synthetically modified mRNA. Using the RNA quantification kit, the encapsulation efficacy of the nanoliposomes can be analyzed after the encapsulation of one microgram of eGFP encoding mRNA by analyzing the free amount of mRNA which is not encapsulated.
After the encapsulation of EGFP mRNA in different amounts of nanoliposomes, the formed nanolipoplexes can be incubated with cells in vitro and the percentage of eGFP-expressing cells can be analyzed using flow cytometry 24 hours post-transfection. As seen here, even just one microliter of the nanoliposomes solution is sufficient to achieve a high transfection of the cells invitro. When the cells are transfected with nanoliposomes containing Cy3 labeled eGFP mRNA, the presence of the eGFP mRNA in the cytoplasm, as well as the already produced eGFP protein, can be visualized.
Since the transfection of cells using nanolipoplexes can have adverse effects on cells, the viability of the cells was tested after 24 hours and 72 hours post-transfection. No effects on cell viability could be detected when the cells were treated with 2.5 or five microliters of nanolipoplexes. When attempting this procedure, it is important to remember to work in an ethanol-free environment all the time because this could harm nanoliposome stability.
While preparing and mixing the DC-cholesterol with DOPE, you should use a glass pipette to avoid contamination of the dissolved lipids with soluble components of plastic pipette tips. Following this protocol, the generated nanoliposomes can be easily manipulated during or after the preparation. For example, incorporation of PEG molecules or specific antibodies would lead to increased stability or initiate active tissue targeting.
The generated nanoliposomes fulfill the necessary safety aspects and facilitate mRNA uptake to the target cells. Nanoliposomes complexed with a specific therapeutic nRNA can make an essential contribution to the development of novel mRNA-based therapeutics in the field of tissue engineering or replacement therapy for example.
