This protocol can be used to produce nucleic acid encapsulated lipid nanoparticles with high reproducibility and speed and low volumes. The formulation parameters can be tuned to achieve a desired bio distribution based on the clinical application. The standard herringbone design of the microfluidic cartridge allows a rapid, precise and controlled laminar flow during mixing.
The method is amenable to scaling up and generates uniform, highly encapsulated particles. With most commercially approved gene therapy products relying on viral methods, this procedure can provide insight into gene delivery, as its non-viral approach allows applications for which repeat dosing is necessary. Begin by adding the appropriate amount of each liquid stock solution to a glass vial with intermittent vortexing.
as indicated in the table, followed by the addition of 200 proof ethanol to a final volume of 533 microliters. Then add the appropriate amount of mRNA stock and dilute with citrate buffer to achieve a final volume of 1.5 milliliters at the desired concentration. To prime the microfluidic channels, first enter the priming parameter into the instrument software as outlined in the table.
Next, open the instrument lid and place a microfluidic cartridge into the rotating block. Load at least 500 microliters of ethanol into a 1 milliliter syringe, taking care of that there are no bubbles or air gaps at the syringe tip and insert the syringe into the right inlet of the cartridge. Load a five milliliter syringe with 1.5 milliliters of citrate buffer, taking care that there are no air bubbles or gaps, and insert the syringe into the left inlet of the cartridge.
Place two 15 milliliter conical tubes into the clip holders to serve as waste containers and click Go"to mix the solutions. When the instrument stops priming as indicated by the bottom blue light shutting off, open the late and properly dispose of the conical tubes and syringes. For lipid nanoparticle formation, set the appropriate formulation parameters as indicated in the table and load a 1 milliliter syringe with a previously prepared lipid mix.
Remove any air gaps or bubbles in the syringe tip and insert the syringe into the right side of the cartridge. Load the previously prepared nucleic acid solution into a 3 milliliter syringe, taking care of that there are no bubbles or air gaps in the syringe tip, and insert the syringe into the left inlet of the cartridge. Place a 15 milliliter RNAase-free conical tube labeled with the sample name into the left tube clip and place a 15 milliliter waste conical in the right tube clip.
Close the instrument lid and click Go.The lipid and mRNA solutions will flow through the microfluidic cartridge and the lipid nanoparticle solution will be collected in the conical tubes. Retain the conical tube at the end of the formulation process, then dilute the lipid nanoparticles with 5 milliliters of PBS and a biological safety cabinet. To perform a buffer exchange, first prewash a 100 kilodalton pore size ultracentrifuge filter with 2 milliliters of PBS, centrifuge, and then empty the PBS from the bottom compartment.
Add the diluted lipid nanoparticles to the top compartment of the pre-washed ultracentrifuge filter for three centrifuge washes, discarding the flow-through and adding 5 milliliters of PBS to the ultracentrifuge filter after the first two washes. After the last wash, pipette the lipid nanoparticle solution against the walls of the ultracentrifuge filter a few times to minimize the nanoparticle loss before transferring the nanoparticle solution to a nuclease-free vial. Then add PBS to bring the nanoparticle suspension to the appropriate experimental concentration and volume as necessary.
To assess the encapsulation efficiency of the lipid nanoparticles, first prepare two-fold serial dilutions of working nucleic acid solution in PBS to generate a standard curve, starting from 500 nanograms per milliliter and making at least five dilutions. Next, prepare the nanoparticle sample dilutions in PBS to achieve an appropriate theoretical concentration that lies around the midpoint of the standard curve. Then prepare the ribo green RNA reagent to detect presence of RNA both inside and outside of the lipid nanoparticle by mixing the appropriate amounts of RNA reagent, Triton X100 and PBS.
Then, to detect the presence of RNA outside of the liquid nanoparticle, mix the appropriate amounts of RNA reagents and PBS only. Load replicates of the nucleic acid standard and lipid nanoparticle solutions into a black fluorescence-capable 96 well plate, then add an equal volume of RNA quantification reagent with, and without Triton X100 to the standard and sample replicates. Shake the plate in the plate reader for five minutes at room temperature protected from light to obtain a thorough mixing of the samples, then measure the fluorescence on a microplate reader at an excitation wavelength of 480 nanometers and an emission wavelength of 520 nanometers.
To measure the hydrodynamic size and poly dispersity of the lipid nanoparticles, first dilute the nanoparticle solution 40 times in PBS and add the solution to a semi micro cuvette. Load the cuvette onto the Zetasizer and select a standard operating procedure to set the instrument to measure the nanoparticles according to their material, dispersant, temperature and cell type. Then click Start"to measure the hydrodynamic size and poly dispersity of the lipid nanoparticles.
To measure the Zeta potential of lipid nanoparticles, dilute the nanoparticle solution 40 times in nuclease-free water and load the solution into a cuvette to the fill line. Load the cuvette into a Zeta potential analyzer, taking care that the electrodes make contact with the instrument, and set the instrument to measure the Zeta potential according to the specific makeup of the lipid nanoparticles. Then click Start"to measure the Zeta lipid nanoparticle potential.
In this analysis, multiple batches of lipid nanoparticles with the same lipid formulation and amine to phosphate ratio were developed on separate days to demonstrate the reproducibility of the technique. As observed, batches one and two exhibited overlapping size distributions with a similar poly dispersity, with no significant differences observed in the size or encapsulation efficiency between the batches. Typically, changes in the formulation parameters induce some small yet statistically significant differences.
For example, decreasing the amine to phosphate ratio results in a 4%decrease in the encapsulation efficiency, with a concomitant increase in the hydrodynamic diameter of the nanoparticles. Lipid nanoparticles formulated with different ionizable lipids but the same amine to phosphate ratio, exhibit a significant change in the encapsulation efficiency as well as slight differences in the particle diameter. The encapsulation of plasmid DNA results in larger particles compared to mRA encapsulating lipid nanoparticles, although both types of nanoparticle demonstrate a similar encapsulation efficiency.
Changes in the flow rate process parameter, however, do not impact the lipid nanoparticle development. Lipid nanoparticles have great applications, the perfect example being the recently approved COVID-19 vaccines. This technique serves as a great tool set and paves the way for future applications by allowing lower limb formulation screening.
