Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

This protocol can be used to formulate consistent lipid nanoparticles encapsulating MRA. Formulation parameters can be tuned and the various lipid nanoparticles can be evaluated for their potency and biodistribution. A standardized protocol for lipid nanoparticle formulation and in vitro and in vivo testing can be used to formulate consistent lipid nanoparticles that can be compared with each other.

There are a lot of small details to keep in mind when formulating and evaluating nanoparticle potency, but which details accounted for? Preparing the formulation should not be difficult. Begin by filling a clean, four liter beaker with 200 to 300 milliliters of fresh 10X PBS, and diluting it to 1X with ultrapure water.

Ensure the final volume of PBS is between two and three liters. Place dialysis cassettes of the appropriate capacity for the lipid nanoparticle, or LNP formulation, into the field beaker. Make the ten-fold dilution of a 100 millimolar citric acid buffer stock using ultrapure water to create a 10 millimolar citric acid buffer for mRNA dilution.

Next, make C 12 200 ionizable lipid, helper lipid, cholesterol and lipid anchored PEG or lipid PEG stock solutions by dissolving each lipid in 100%ethanol. Ensure the stock solutions are fully dissolved by heating and mixing them at 37 degrees Celsius. Combine the calculated volumes of the different lipid components in a conical tube.

Dilute the lipids with 100%ethanol to final volume of V, which represents 25%of the final volume of LNP. Calculate the amount of mRNA required for formulation based on the ionizable lipid to mRNA weight ratio and the total volume of mRNA-LNP. Next, thaw the firefly luciferase encoding mRNA on ice.

Then dilute the mRNA to a final volume three times the organic phase using the 10 millimolar citric acid buffer. Spray a delicate task wipe with a surface decontaminate for RNAs and DNA, and wipe the interior of the microfluidic instrument thoroughly. Next, open a new microfluidic cartridge and insert it with the inlet channels facing down and away.

Arrange two sterile 15 milliliter conical tubes on the holder with the caps off. Once done, fill a five milliliter syringe with the mRNA solution containing mRNA diluted in 10 millimolar citric acid buffer. Then fill a three milliliter syringe with the lipid solution containing the lipids diluted in 100%ethanol.

Ensure to remove any air from the syringes. Then without the needle, insert the five milliliter syringe containing the mRNA solution into the left inlet of the cartridge, and insert the three milliliter syringe containing the lipid solution into the right inlet of the cartridge. Turn on the microfluidic instrument and select quick run.

After selecting the correct syringe types for the five and three milliliter syringes inserted, input the parameters for LNP formulation at a three to one aqueous to organic flow rate ratio, before pressing the start button to formulate the mRNA-LNPs. Load the mRNA-LNPs collected in the right conical tube into the previously hydrated dialysis cassettes using a syringe without puncturing or touching the cassette membrane, place the dialysis cassettes containing the mRNA-LNPs back into the beaker containing PBS and let them dialyze for at least two hours. After dialysis, bring the dialysis cassettes containing the mRNA-LNPs into a sterile biosafety cabinet and remove the mRNA-LNPs using a syringe with a needle.

Sterile filter the mRNA-LNPs using a 0.22 micrometer syringe filter and collect them in a sterile conical tube. Then place 65 microliters of the mRNA-LNP solution into a 1.5 milliliter microtube for characterization. Plate 5, 000 HepG2 cells in a white wall, clear bottom, 96-well plate in 100 microliters of complete cell culture medium and incubate the cells overnight in a controlled incubator at 37 degrees Celsius containing 5%carbon dioxide.

Before treatment of cells with mRNA-LNPs, remove the complete medium from the wells. Next, treat the cells with either 100 microliters of mRNA-LNPs diluted in complete cell culture medium at the desired doses, or 100 microliters of complete medium before a 24 hour incubation. After incubation, remove the cell culture medium from the 96-well plate, add 20 microliters of lysis buffer to each well followed by 100 microliters of the luciferase assay reagents.

Protect the plate from light and place the plate into a plate reader to measure the bioluminescent signal. Dilute the mRNA-LNP solution using sterile PBS so that two micrograms of total mRNA is present in 100 microliters of the tail vein injection volume. Fill a 29 gauge insulin syringe with either 100 microliters of mRNA-LNP or 100 microliters of PBS and remove any bubbles from the syringe.

Insert the needle with the bevel facing up into the mouse's lateral tail vein and slowly inject the 100 microliters of mRNA-LNP or PBS. Six hours post injection, prepare a 15 milligram per milliliter stock solution of D-Luciferin potassium salt in sterile PBS. In the imaging software for the in vivo imaging system, or IVIS, select the box next to luminescence before pressing initialize to prepare the instrument for data acquisition.

Next, administer 200 microliters of D-Luciferin intraperitonealy to the previously treated mice and wait for 10 minutes while the luciferase signal stabilizes before anesthetizing the mice. Transfer the anesthetized mice to the nose cones inside the in vivo imaging system, ensuring that they're on their backs with their abdomens exposed, before closing the chamber door. In the imaging software, ensure that the correct field of view is selected and set the exposure time to auto, click the acquire button in the software to obtain bioluminescence images.

The encapsulation efficiency of the mRNA-LNPs was determined as 92.3%using the modified fluorescence assay and equation shown here. Increasing bioluminescence was observed upon treatment of 5, 000 HepG2 cells with increasing doses of mRNA-LNP. Luciferase expression was easily detected at this study's lowest dose of five nanograms per well.

A strong bioluminescence signal was observed in the upper abdomen of the mice treated with formulated LNPs. Regions of interest were drawn around the liver signals to calculate the total luminescent flux. The total luminescent flux was approximately five times 10 to the 9th.

Always maintain the ionizable lipid to nucleic acid weight ratio when formulating LNPs. Every volume of the organic phase should contain ionizable lipid corresponding to mRNA in three volumes of the aqueous phase.