Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (µMIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

Our research is focused on understanding nanoparticle formation through turbulent mixing of solvent and antisolvent streams in confined geometries. Confined turbulent mixing enables reproducible production of lipid nanoparticles under continuous flow conditions ranging from laboratory scale at hundreds of microliters to industrial scale at dozens of liters per minute. Lipid nanoparticles are typically prepared with microfluidic devices or by pipette mixing.

These microfluidic devices mix ethanol solvent and aqueous antisolvent streams in these capillary-like channels. However, these small channels and low flow rates yield a low Reynolds number, which leads to laminar mixing. And pipette mixing produces LNPs by injecting the solvent stream directly into an antisolvent bath.

The current methods to make lipid nanoparticles have poor scalability and reproducibility. For example, pipette mixing is inherently variable due to the unconfined manual mixing. While microfluidic devices address some of these issues, they operate at very low flow rates and they foul due to lipid RNA deposition on the channel walls.

These issues highlight the need to have reproducible techniques with high throughput at laboratory to clinical scales. This protocol demonstrates reproducible and scalable production of lipid nanoparticles through turbulent mixing across different batch sizes. Researchers can confidently perform LNP formulation screening or optimization at small scales before producing larger batches of materials for extended trials.

Turbulent mixing ensures consistent nanoparticles, regardless of the batch size. Confined geometry turbulent mixers address the major issues with existing lipid nanoparticle production techniques. Using turbulent conditions produces faster, molecular-scale mixing, which enables the incorporation of differently-sized oligonucleotides into the LNP core.

The CIJ mixer also eliminates the fouling caused by lipid deposition on the mixer walls. Turbulent flows at the same Reynolds number have self-similar turbulent Kolmogorov scale vortices for consistent mixing. 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 ethanolic 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.

The multiple inlet vortex mixer has tangentially injected streams into the mixing chamber that form lamellae in the rotating vortex. A high Reynolds number in the mixing chamber ensures the production of micron scale turbulent vortices, which rapidly mix the lamellae, similar to the turbulent vortices inside a CIJ mixer. To begin assembling the micro-size-multi-inlet vortex mixer, or MIVM, gather the required bottom receiver, the mixing geometry disc, the top disc, the O-ring, and the spanner wrench on a working platform.

Seat the O-ring into the groove in the mixing disc. Align the mixing disc holes with the pegs on the top disc and push them together without dislodging the O-ring. Screw the mated mixing disc O-ring and top disc assembly into the bottom receiver loosely.

Then, tighten the top disc into the bottom receiver, using the spanner wrench. Insert and tighten the outlet tubing fitting into the bottom receiver to complete the MIVM. Mount the assembled MIVM into the mixer stand so that the outlet tubing exits through the support plate.

Next, prepare the solvent and antisolvent solutions in the desired composition. Load 30 milliliters of solutions into gas-tight syringes. Invert the syringe and tubing and expel any air from them.

Attach PTFE tubing with a Luer adapter fitted on the end. Mount the syringes into a syringe pump and attach them to the mixer inlets on the MIVM. Dilute the 100 millimolar pH 4 acetate buffer stock to 10 millimolar in a total volume of 320 milliliters.

On the syringe pump, set the volumetric flow rate to 20 milliliters per minute. Start the syringe pump and allow the first 10 seconds of effluent to flow into a waste beaker. After 10 seconds, collect the MIVM effluent in the quench bath.

Now, remove and cap the quench bath containing lipid nanoparticle dispersion. Clean the MIVM between each experiment by flushing out at least twice the volume of outlet tubing. After detaching the mixer from the stand, while keeping the syringes attached, hold it over a waste container.

Remove the syringes, letting the holdup volume drain into the container. Then, hold the mixer assembly upside down and use the spanner wrench to disassemble the mixer. Rinse the outlet tubing and mixing geometry with ethanol and dry the components using a stream of air or nitrogen.

Then, rinse the O-ring with deionized water and blot dry. Rinse the top disc and syringe thoroughly with ethanol before using an air or nitrogen stream to dry the surface.