The confined impinging jet mixer directs two high-Reynolds number streams at each other inside a larger mixing chamber. The linear momentum of each jet is dissipated in the formation of micron-scale turbulent vortices that cause rapid and uniform mixing of the solvent and antisolvent streams. To begin formulating the lipid nanoparticles, fill two five-milliliter syringes with ethanol and lock each into an inlet port of the confined impinging jet, or CIJ mixer.
Rapidly depress the syringes and collect the mixer effluent as waste. Remove the ethanol-flush syringes from the CIJ. Blow a dry nitrogen stream through the inlet adapters to dry the internal channels of the CIJ.
Mix the lipid stock solutions and dilute them with ethanol in a 1.5-milliliter microcentrifuge tube. Dilute 100-millimolar pH 4 acetate buffer stock to 10 millimolar in a total volume of four milliliters. To prepare antisolvent stream, mix RNA with acetate buffer in a 1.5-milliliter tube.
Prepare 500 microliters of solution in a one-milliliter syringe. Invert the syringe and expel any air from the aqueous antisolvent stream. Similarly, collect 500 microliters of lipid solution in a one-milliliter syringe.
Then, invert the syringe to expel air from the ethanoic solvent stream. Position the clean CIJ mixer over the quench bath vial. Mate the two syringes to the CIJ mixer inlet ports.
Depress both syringes rapidly to mix the solvent and antisolvent streams and collect lipid nanoparticles in the quench bath. Remove the CIJ mixer from the quench bath with the syringes still attached. Hold the CIJ over a waste container and remove the syringes, allowing the residual holdup volume to flow into the waste container.
Now cap the quench bath containing lipid nanoparticle dispersion and gently swirl the vial.
