Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

Summary

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.

Abstract

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.

Introduction

mRNA-based therapeutics hold great potential for the treatment and prevention of a wide range of diseases, including infectious diseases, genetic disorders, and cancers¹. Unlike small-molecule therapeutics, which can passively diffuse across the cell membrane, nucleic acids must be encapsulated for intracellular delivery². Encapsulation provides both structure and stability to mRNA, facilitating their intracellular delivery via endocytic pathways as well as preventing degradation from intra- and extra-cellular components such as nucleases³. A host of materials and nanocarriers have been developed for the encapsulation and delivery of mRNA, including inorganic nanoparticles, polymers, lipids, and lipid-like materials¹. Among these, LNPs have emerged as the most prominent delivery platform for mRNA-based therapeutics⁴.

LNPs are composed of four lipid components: ionizable lipid, cholesterol, zwitterionic lipid, and PEG-lipid stabilizer⁵. Ionizable lipids suitable for mRNA delivery exhibit a careful balance between the lipid hydrophobicity and the dissociation constant (pKa) of a ternary amine group⁶. The ionizable lipid pKa typically has a pH between 6.0 and 6.7, such as KC-2 (DLin-KC2-DMA), MC-3 (DLin-MC3-DMA), and ALC-0315⁷. This pKa restriction on the ionizable lipid enables both the encapsulation of nucleic acid polymers as hydrophobic lipid salts and the intracellular delivery through an "endosome escape" process. LNPs enter a target cell through (various) endocytosis pathways that all involve acidification of the endosome from pH 7.4 to pH ~5⁸. The ionizable lipid pKa ensures that LNPs have nearly neutral surfaces under physiological conditions but become cationic in an acidifying endosome⁹. This pH response enables selective disruption of only the endosomal membrane, the release of the encapsulated nucleic acid polymer, and preserves cell viability, unlike permanently cationic lipids used in transfection systems such as Lipofectamine. Cholesterol is a hydrophobic, interstitial molecule in the LNP structure that improves lipid fluidity. The zwitterionic lipid plays a structural role and forms a bilayer on the LNP surface. The poly(ethylene glycol)-lipid (PEG-lipid) is a colloidal stabilizer that enhances the LNP stability by imparting a polymeric steric stabilizer onto the LNP surface, which resists aggregation of LNPs. This stabilizes the LNP, particularly during changes in pH that regenerate the free base form of the ionizable lipid which behaves like a hydrophobic oil. The Onpattro (patisiran) recipe (hereafter, referred to as LNP formulation) is often used as a starting point for LNP formulation with ionizable lipid MC3, cholesterol, distearoylphosphatidylcholine (DSPC), and PEG2000-DMG dissolved in ethanol mixed against an aqueous solution of RNA¹⁰.

Several techniques can be used to manufacture LNPs encapsulating nucleic acid polymers, with most of them relying on a common theme of rapidly mixing an ethanol stream containing lipids with an aqueous stream incorporating the nucleic acid of interest (siRNA, mRNA, or DNA)^{9,11,12,13,14}. In this regard, bulk mixing processes such as pipette mixing and vortex mixing offer a simple strategy to form LNPs that eliminates the need for using sophisticated instruments¹². However, bulk mixing does not provide a homogeneous distribution of components, leading to a sub-optimal LNP size distribution along with significant batch-to-batch variability¹⁵.

Laboratories routinely use microfluidic mixing techniques to obtain reproducible LNPs by achieving more precise control over mixing conditions^12,13,16. Yet, the laminar flow conditions in microfluidic devices, which are inherent due to the small length scales and low velocities in a microfluidics chamber, result in comparatively slow solvent/antisolvent mixing¹⁷. The small chamber dimensions severely limit the throughput and scalability needed for GMP production of LNPs, but researchers have parallelized microfluidic chambers to attempt to scale production volumes¹⁵. A parallelized microfluidics geometry does not eliminate the problem of lipid adsorption to surfaces during large volume processing, a problem commonly referred to as "fouling" of the mixing device, and there are problems with uniformity and stability of flows that make microfluidics scaleup challenging for industrial-scale production^18,19. It is not surprising that pharmaceutical companies used turbulent impinging jet mixers to manufacture COVID-19 vaccination mRNA-LNPs²⁰.

The production process of RNA-loaded LNPs entails the blending of an aqueous buffer stream containing the RNA payload with an ethanol stream containing the four distinct lipid components. These formulations utilize an acidic buffer with a pH of 4.0 or less, which charges the ionizable lipid as the aqueous and ethanolic streams mix. The positively charged ionizable lipids interact electrostatically with the negatively charged RNAs, forming a hydrophobic RNA-lipid salt. Hydrophobic lipid species, including the RNA-lipid salt, precipitate in the mixed solvents and form hydrophobic nuclei. These nuclei grow through the precipitation of zwitterionic lipid and cholesterol until reaching a critical point where sufficient pegylated lipid adsorbs on the surface of the LNPs, halting further growth -nucleation and growth mechanism^21,22,23. The addition of aqueous buffer to the lipid solution, to the extent where lipids precipitate and LNPs form, hinges on two distinct time scales: the solvent-antisolvent mixing period, τ_mix, and the nuclei growth period, τ_agg. The dimensionless Damköhler number, defined as Da = τ_mix/τ_agg, captures the interplay between these time scales²⁴. In instances of slow mixing (Da > 1), the final size of LNPs is transport-controlled and varies with mixing time. Conversely, during fast mixing (Da < 1), the fluid is fragmented into Kolmogrov-length striations or layers, whereby LNP formation is solely governed by the molecular diffusion of each constituent, resulting in homogeneous kinetics of LNP formation. Achieving the latter scenario demands that the lipid concentration exceeds a critical threshold, establishing a state of supersaturation conducive to uniform homogeneous nucleation.

It is estimated that τ_agg ranges from a few tens to a few hundreds of milliseconds²⁵. In its most basic configuration, the two streams, one containing ethanol with lipids and the other containing an aqueous buffer with RNA cargo, are injected into a chamber known as a "confined impinging jet" (CIJ) mixer. Turbulent vortices produce solvent/antisolvent striation length scales of 1 µm within 1.5 ms when operated at appropriate velocities. The stream velocities and mixing geometry determine the conversion of linear momentum into turbulent vortices that mix the streams. This is parameterized by the dimensionless number, the Reynolds number (Re), that is linearly proportional to the flow velocities. Re is computed from Re = Σ (V_iD_i/v_i), where V_i is the flow velocity in each steam, v_i is the kinematic viscosity of each stream, and D_i is the stream inlet diameter in 2-jet CIJ devices²⁶ or the chamber diameter in 4-jet MIVMs²⁷. Note: Some references for the CIJ use only a single jet diameter and velocity to define Re²⁸. Re is in the range of 1-100 in a microfluidics device, whereas in the CIJ devices, Re of 125,000 can be achieved. In a CIJ mixer, streams with equal momentum collide, dissipating their momentum upon impact as turbulent mixing, which leads to efficient micromixing due to the small Kolmogorov microscales and small Damköhler number. Another type of mixer is the "multiple inlet vortex mixer" (MIVM), where four streams are directed into a central chamber. In this setup, continuous flows into the confined mixing chamber ensure a well-defined mixing time scale. All fluid elements pass through the high-energy mixing zone in both types of mixers. In contrast, simple mixing devices like T-junctions do not contain a chamber that provides a mixing zone, resulting in less mixing of the two streams due to the incoming stream momentum being largely deflected into the outlet direction rather than into turbulent vortex generation. Both CIJ and MIVM mixers can be operated in batch or continuous modes, offering flexibility for LNP production at various scales.

This protocol describes how optimal LNP formulations are made by employing two confined impinging jet technologies: 2-jet CIJ and the 4-jet MIVM mixers. The operation of CIJ and MIVM mixers has been previously demonstrated for the preparation of NPs with hydrophobic core materials²⁹. That article and video should be consulted as an additional resource on the formation of NPs with these mixers. This update focuses on lipid-based NP formation. The ability to tune the size of LNPs by varying the micro-mixing conditions is demonstrated. Additionally, the utility of CIJ technologies in forming stable, monodisperse LNPs with improved in vitro transfection efficiencies in HeLa cells when compared to LNPs made using poor pipette mixing is shown. Furthermore, the advantages and disadvantages of each CIJ mixing geometry, along with appropriate conditions needed for the scaleup of these mixers, are discussed.

Protocol

The details of the reagents and the equipment used in this study are listed in the Table of Materials.

1. Preparation of buffers, solvent and antisolvent streams

1. Dissolve all lipid components in ethanol at given mass concentrations, as shown in Table 1, to make a patisiran LNP formulation¹⁰. Prior to use, heat the solutions to 37 °C with gentle sonication to re-dissolve any precipitated solids.
   NOTE: Larger stock solutions of several milliliters can be prepared and stored at 4 °C.
2. Prepare an acetate buffer stock solution at a concentration of 100 mM, pH 4, by combining 186 mg of sodium acetate and 464 mg of acetic acid in 80 mL of nuclease-free water. Adjust the pH with concentrated HCl or NaOH as needed and make up the final volume to 100 mL.
3. Prepare a HEPES buffer stock solution at a concentration of 1 M, pH 7.5, by dissolving 23.82 g of HEPES salt in 80 mL of nuclease-free water. Adjust the pH with concentrated HCl or NaOH as needed and make up the final volume to 100 mL.
4. Dilute the nucleic acid polymer of interest and the 100 mM acetate buffer stock with nuclease-free water to produce a 300 µg/mL solution of RNA in 30 mM acetate buffer. Prepare an adequate volume of working solution for the designed experiments, and this protocol requires 1500 µL total for both the CIJ and MIVM mixers.
   NOTE: RNA obtained from the supplier will already be in an appropriate buffer, typically at a concentration of 10 mg/mL (yeast RNA) or 1 mg/mL (luciferase-encoding mRNA, LucRNA, or GFP-encoding mRNA, GFP-RNA). Yeast RNA may be used as a low-cost model RNA for precipitations.

2. Formulation of LNPs using a two-jet CIJ mixer

1. Preparation and cleaning of equipment
   1. Clean CIJ mixers immediately before use by flushing the mixer with ethanol. Fill two 5 mL syringes with ethanol and lock each into an inlet port of the CIJ. Rapidly depress the syringes and collect the mixer effluent as waste.
      NOTE: CIJ mixers may be constructed and operated as previously described²⁹. Various support stands may be used to hold the CIJ mixer, including a ring stand, centrifuge tube holder, or Erlenmeyer flask. The CIJ suppliers are listed in the Table of Materials and the Supplementary File 1.
   2. Remove the ethanol flush syringes from the CIJ. These syringes may be stored and reused for additional ethanol rinses of the CIJ.
   3. Dry the internal channels of CIJ by blowing a dry nitrogen stream through the inlet adapters. Use only dry, filtered air that is not contaminated with pump oil mist or aerosols if nitrogen is not available.
2. Preparation of solvent and antisolvent streams
   1. Mix the lipid stock solutions and dilute with additional ethanol to produce 500 µL of the 6 mg/mL lipid solution listed in Table 1 in a 1.5 mL microcentrifuge tube.
      NOTE: This formulation is the commonly used Onpattro composition containing 50 mole% MC3, 10 mole% DSPC, 38.5 mole% cholesterol, and 1.5 mole% DMG-PEG2000.
   2. Dilute the 100 mM pH 4 acetate buffer stock to 10 mM in a total volume of 4 mL as the CIJ mixer effluent quench bath.
      NOTE: A magnetic stir bar may also be added to the quench bath.
   3. Withdraw 500 µL of RNA solution prepared in step 1.4 in a 1 mL syringe. Invert the syringe and expel any air from this aqueous antisolvent stream containing nucleic acid polymer.
   4. Withdraw 500 µL of the lipid solution prepared in step 2.2.1 in a 1 mL syringe. Invert the syringe and expel any air from this ethanolic solvent stream containing lipids.
3. LNP production in a CIJ mixer
   1. Position the clean CIJ mixer over the quench bath vial.
      NOTE: A ring stand clamp or tube rack makes a convenient support for the CIJ mixer. See Figure 1A for a typical CIJ setup.
   2. Mate the two syringes filled in step 2.2.3 and step 2.2.4 to the CIJ mixer inlet ports.
   3. Depress both syringes rapidly to mix the solvent and antisolvent streams and collect lipid NPs in the quench bath.
      NOTE: the syringes must be advanced quickly (in under 1 s) but smoothly and uniformly. Asymmetric flow or slow flow will produce polydisperse, large LNPs.
   4. Remove the CIJ mixer from over the quench bath with the syringes still attached.
      NOTE: Do not remove the syringes or allow the hold-up volume to flow into the quench bath, as this material is poorly mixed and will adversely impact the production dispersion properties if mixed into the quench bath.
   5. Hold the CIJ over a waste container and remove the syringes, allowing the residual hold-up volume to flow into the waste container. Dispose these syringes and repeat the cleaning process as described in section 2.1.
   6. Analyze LNPs produced with the CIJ as described in section 5 below.

3. Formulation of LNPs using a four-jet MIVM mixer

1. Assembly of a MIVM mixer, referred to as a micro-size MIVM (µMIVM) to distinguish it from larger models used for scaled-up production
   1. Gather all the individual components required to assemble a MIVM mixer: the bottom receiver, the mixing geometry disk, the top disk, the O-ring, and the spanner wrench.
      NOTE: MIVM construction, assembly, and operation have been previously demonstrated for the encapsulation of hydrophobic species, and the suppliers are listed in Supplementary File 1 and the Table of Materials²⁹. See Figure 2 for a schematic of components and mixer stand terminology.
   2. Seat the O-ring into the groove in the mixing disk.
   3. Align the mixing disk holes with the pegs on the top disk and push them together without dislodging the O-ring.
   4. Screw the mated mixing disk, O-ring, and top disk assembly into the bottom receiver loosely.
      NOTE: Remove the outlet tubing from the bottom receiver before this step.
   5. Tighten the top disk into the bottom receiver using the spanner wrench.
      NOTE: A food or pharmaceutical-grade anti-seize compound may be applied to the top disk threads if thread galling is observed.
   6. Insert and tighten the outlet tubing fitting into the bottom receiver to complete the MIVM.
   7. Mount the assembled MIVM into the mixer stand so the outlet tubing exits through the support plate.
      ​NOTE: The procedure for adjusting the MIVM mixer stand should be periodically performed to make sure the mechanical stops on the mixer stand are correctly configured²⁹.
2. Preparation of solvent and antisolvent streams
   1. Mix the two ethanolic lipid solvent stream solutions in a 1.5 mL microcentrifuge tube, as given in Table 2, by diluting the lipid stock solutions and adding additional ethanol as needed to reach a final lipid concentration of 6 mg/mL.
      NOTE: This is the same lipid solution composition as in 2.2.1 but with a total volume of 1000 µL.
   2. Dilute the 100 mM pH 4 acetate buffer stock to 10 mM in a total volume of 8 mL as the MIVM effluent quench bath.
      NOTE: A magnetic stir bar may also be added to the quench bath.
3. Formulation of LNPs using a MIVM mixer
   1. Place the 8 mL quench bath underneath the mixer stand so that the outflow tubing is emptying into the quench bath.
   2. Draw up the solvent and antisolvent streams into 1 mL gas-tight syringes using a blunt tip needle. Remove all air bubbles and dispose of the needle. Prime each syringe by inverting and expelling any air from the syringe.
   3. Assemble the four syringes onto the mixer in a clockwise fashion (Syringes 1-4, Table 2) with the same streams on the opposite sides. See Figure 1B for the final appearance schematic.
   4. Hold the bearing housing on both sides of the mobile plate. Avoid placing the fingers on the bottom face of the housing, as this poses a pinch hazard due to the mechanical stops. Carefully lower the mobile plate until it evenly rests on the syringes.
      NOTE: The syringe plungers must all be at an equal height before operation.
   5. Steadily and smoothly depress the plate, aiming to complete the operation in about 0.5 s to 1 s for these stream volumes.
   6. Cap the quench bath, which contains the LNP dispersion.
   7. Perform the cleaning of the MIVM after use, following the instructions in step 3.5 below.
4. Formulation of LNPs using a MIVM mixer driven by a syringe pump
   1. Assemble the MIVM as described in step 3.1.
   2. Prepare the solvent and antisolvent solutions in the desired composition and at a sufficient volume for the required formulation size (Table 3).
      NOTE: These are the same compositions as the aqueous antisolvent (step 1.4) and ethanolic solvent (step 3.2.1) streams but are scaled up for use with the larger syringe volumes in Table 3.
   3. Load the solutions into gas-tight syringes with 20 mL volumes and attach PTFE tubing with a luer adapter fitted on the end. Prime the syringes and tubing by inverting the syringe and tubing and then expelling any air.
   4. Mount the syringes into a syringe pump and attach the syringes to the mixer inlets on the MIVM, as shown in Figure 3A.
      NOTE: The CIJ may also be operated via pumps in the same way but with only one antisolvent and solvent stream syringe.
   5. Dilute the 100 mM pH 4 acetate buffer stock to 10 mM in a total volume of 320 mL as the MIVM effluent quench bath.
   6. Place the quench bath underneath the MIVM outlet.
   7. Set the volumetric flow rate on the syringe pump to 20 mL/min.
      NOTE: The volumetric flow rates may be varied from 2 mL/min to 40 mL/min by manually setting the values on the syringe pump to form LNPs with different micromixing conditions.
   8. Start the syringe pump, but allow the first 10 s of effluent to flow into a waste beaker. Collect the MIVM effluent in the quench bath after this 10 s start-up flow period.
   9. Remove and cap the quench bath containing LNP dispersion made at the selected (20 mL/min) volumetric flow rate.
      NOTE: This procedure may be repeated to synthesize LNPs at different flow rates by selecting appropriate quench bath volumes.
   10. Clean the MIVM between each experiment (when changing the flow rate) by flushing out at least twice the volume of outlet tubing to prevent collection of LNPs made at the previous flow rate condition before beginning the next LNP synthesis.
5. Equipment cleaning after use
   1. Detach the mixer from the stand while keeping the syringes attached, and hold it over a waste container. Remove the syringes, letting the hold-up volume drain into the container. Then, hold the mixer assembly upside down and use the spanner wrench to disassemble the mixer.
   2. Rinse the outlet tubing with solvent (e.g., ethanol) and dry with air or nitrogen.
   3. Rinse the mixing geometry with a suitable solvent, such as deionized water or ethanol, followed by a rinse with ethanol. Dry the components using a stream of air or nitrogen.
   4. Rinse the O-ring with deionized water and blot dry.
      NOTE: If the O-ring looks stretched or deformed, let it air dry overnight before using it. Maintain a large stock of O-rings since they are consumable parts. If the shape does not recover by the next day, dispose of the O-ring.
   5. Rinse the top disk thoroughly with solvent, using an air or nitrogen stream to dry the surface and the syringe fittings.
   6. Rinse each syringe with a good solvent (e.g., deionized water or ethanol). Apply a final rinse with ethanol and air dry before the next use.

4. Post-processing of LNPs

1. Buffer exchange and ethanol removal
   1. Dialyze the LNP dispersion against a 10 mM HEPES buffer at pH 7.4 utilizing an appropriately sized dialysis cassette with a 20 kDa molecular weight cutoff dialysis cartridge.
      NOTE: This step both removes the residual 10% ethanol and increases the dispersion pH by replacing the acetate buffer with HEPES.
   2. Dilute the stock 1 M HEPES buffer to 10 mM in a total volume of 1 L.
   3. Load the LNP dispersion into a 12 mL dialysis cassette using a syringe and needle.
   4. Immerse the dialysis cartridge in the 1 L of HEPES buffer and magnetically stir the external buffer. Change the external HEPES buffer after 3 h, and remove the dialysis cartridge after an additional 3 h for a total dialysis time of 6 h.
   5. Withdraw the dialyzed LNP dispersion from the dialysis cartridge and discard the spent cartridge.
2. LNP suspension concentration via ultracentrifugation
   1. Pipette the suspension into an appropriately sized centrifugal filter with a MWCO of 100kDa³⁰.
      NOTE: Centrifugal filters are available in a range of sizes, commonly from 0.5 mL to 12 mL.
   2. Centrifuge the dispersion at 2000 x g for 10-20 min (at room temperature) to increase the concentration by a factor of approximately 5-20x.
      NOTE: During centrifugation, check on the sample every 5 min to ensure that an appropriate amount of liquid remains.

5. Characterization of LNPs

1. Hydrodynamic diameter measurements with dynamic light scattering (DLS)
   1. Dispense 750 µL or 900 µL of the prepared LNP dispersion (without any dilutions) into a plastic micro or square cuvette, respectively.
      NOTE: Smaller volumes can also be used in appropriate cuvettes.
   2. Set the appropriate viscosity for the solvent in which the LNPs are dispersed (i.e., 1.26 cP for 10 vol% ethanol mixture).
   3. Collect backscattered light at a 173° angle at 25 °C, followed by the size determination of LNP using the Stokes-Einstein model and the first cumulant of the light scattering correlation function series expansion as defined in the ISO standard document 13321:1996 E.
   4. Repeat the measurements at least three times.
2. Zeta potential measurements
   1. Add ~800 µL of as-prepared LNP dispersion into folded capillary zeta-sizer cells.
      NOTE: To avoid bubble entrapment within the capillary channel, half of the liquid is dispensed into the cells, held upside down, and then rotated.
   2. Repeat the measurements at least three times.
      NOTE: Buffer conductivity should be between 0.2-2 milliSiemens/cm for best results.
3. Encapsulation efficiency (EE) measurements by commercially available RNA quantification kit
   1. Dilute the appropriate amount of the 20x Tris-EDTA (TE) buffer at pH 7.5 provided in the assay kit to 1x concentration using nuclease-free water.
   2. Prepare a Triton X-100 solution at 2 wt%.
   3. Dilute the appropriate amount of LNP dispersions in the 1x TE buffer to yield a total mass concentration of approximately 0.6 µg/mL for RNA, with ethanol content below 0.2 vol%.
   4. Prepare similar samples as in the previous step but containing 0.5 wt.% Triton X-100, which effectively dissolves the LNPs, facilitating the differentiation between "free" and total RNA concentrations in the absence and presence of Triton X-100, respectively.
   5. Prepare appropriate amounts of control RNA solutions at the concentrations of 0 µg/mL, 0.1 µg/mL, 0.2 µg/mL, 0.4 µg/mL, 0.6 µg/mL, 0.8 µg/mL and 1.0 µg/mL in the 1x TE buffer.
      NOTE: The RNA kit may initially need to be diluted.
   6. Repeat the previous step but at 0.5 wt.% Triton X-100.
   7. Dilute the Ribogreen dye (provided in the kit) 200 times in the 1x TE buffer.
   8. Add appropriate volumes of the diluted Ribogreen dye to all prepared control RNA and sample solutions, whether with or without Triton X-100. The dye should be diluted equally across both control RNA and sample solutions at a factor of two. Consequently, the total dilution of the dye is 400 times that of its original concentration in the assay kit.
   9. Vortex mix the containers.
   10. Prepare a 96-well, non-treated, flat-bottom, opaque plate for analysis in a plate reader.
   11. Dispense 100 µL volumes of samples and RNA controls into separate cells of the plate.
       NOTE: Watch for bubble formation in solutions containing Triton X-100. At least three replicates are conducted for each sample, requiring at least 350 µL of samples.
   12. Record fluorescence intensity using the plate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm, with an illumination duration of approximately 10 s per reading. No shaking is needed.
       NOTE: The EE is determined from , where I_{with Triton} and I_{without Triton} represent the averaged fluorescence intensity of three replicates of samples with and without Triton X-100, respectively, and a_{with Triton} and a_{without Triton} denote the slope of linear fits to the two averaged calibration curves with and without Triton X-100, respectively³¹.

Discussion

Synthesis of LNPs containing nucleic acid polymers using two confined impinging jet turbulent mixers has been presented. When conducted at appropriate velocities, CIJ turbulent mixers ensure the time scale of mixing is shorter than the LNP assembly time, producing homogenous supersaturation conditions for forming small LNPs with narrow size distributions²¹. Consequently, LNPs made with the same chemistry using different turbulent mixer geometries (the 2-jet CIJ and the 4-jet MIVM mixer) exhibit similar physicochemical properties and show good transfection efficiencies (Figure 5 and Figure 6). In contrast, LNPs made using pipetting that produces poorer mixing results in larger and more polydisperse LNPs (Figure 5A) with lower transfection efficiencies. It has long been understood that mixing and assembly kinetics play a significant role in LNP processing; Cullis et al. noted that rapid convective-diffusive mixing of ethanol and buffer leads to the formation of small particles with a narrow size distribution, whereas slow diffusive mixing leads to larger particles with broad size distributions⁹. The time scale of mixing in CIJ turbulent mixers decreases proportionately to the inlet flow rates of the streams to the mixer²⁷. This is quantified by the dimensionless Reynolds number (Re), which measures the ratio between the inertial and viscous forces. The turbulence inside the mixing chambers of the CIJ and MIVM occurs at sufficiently high Re, such that the turbulent vortex stretching results in small scales of length that produce rapid solvent/antisolvent mixing by diffusion. The turbulent length scale depends on the Re and not the specific geometry of the mixing device. That is why either the CIJ or the MIVM makes the same LNP particles, and why various sizes of MIVM mixers make the same NP sizes²⁷. At high Re, corresponding to high inlet velocities, LNPs can be made reproducibly without batch-to-batch variations (Figure 3B).

This protocol enables the formulation of a variety of mRNA, DNA, or siRNA LNPs with different physicochemical properties using turbulent CIJ mixers. In addition to allowing versatility in composition and concentrations, this technique provides a clear path to rapidly screen formulations at bench sale (a few milligrams) and scale up the lead formulations to larger industrial batch sizes at production rates of 5 L/min³⁶. This has been a major hurdle for several other techniques, including bulk-mixing and microfluidics. For instance, bulk-processing techniques fail to consistently manufacture LNPs reproducibly, even at a few milliliter scales. Microfluidic techniques provide a significant improvement over bulk-mixing techniques to enable the production of uniform and reproducible LNPs; however, they are only in the milligram range²⁹. As detailed in the introduction, parallelization of microfluidic devices provides an attempt at scaling to production scales but does not eliminate the issue of fouling, and it cannot be scaled as successfully as mixers based on confined impinging jet technology.

Apart from these advantages, CIJ mixers will be instrumental in manufacturing next-generation LNPs that exhibit targeting capabilities or perform gene editing. The current LNP formulations have lipids and nucleic acids that have similar diffusivities, and therefore, they can be made even with slightly poor mixing at bench-scale. However, gene-editing approaches may require the encapsulation of nucleic acid species with widely different molecular weights, such as small guide RNA molecules and large mRNA transcripts, to code a CAS9 protein³⁷. The very different diffusion time scales of these different species make uniform encapsulation at stoichiometric ratios challenging. This problem of uniform encapsulation becomes more pronounced as the mixing efficiency becomes poorer. Likewise, targeting non-hepatic cells might need the incorporation of strongly bound slow-diffusing stabilizers (such as large molecular weight block copolymers with targeting ligands). Targeting ligands as large as 14 kDa can be conjugated to block copolymers prior to nanoparticle assembly, which enables their uniform incorporation into NPs using CIJ mixing³⁸. CIJ turbulent mixers are useful tools for manufacturing LNPs made with components having different diffusivities.

While CIJ turbulent mixers demonstrate several advantages over other mixers for formulating LNPs, it is important to note the limitations associated with each geometry. The 2-jet CIJ mixer requires that both the inlet streams (ethanol and water) have equal momenta (within 10%-30%) to achieve uniform turbulent micromixing in the chamber. The fact that the exit stream comprises 50:50 solvent/antisolvent limits the level of supersaturation in the mixing cavity where precipitation occurs²⁹. This drawback is addressed by the 4-jet MIVM mixer, as it can utilize four jets with unequal momenta to accomplish high supersaturation conditions in the mixing chamber. Additionally, both the mixers are required to be in the order of milligrams of total mass, making them a non-ideal choice for high-throughput screening of many different LNP formulations. For simple LNP formulations, screening may best be done with microfluidics or pipetting strategies at microgram scales and then transferred to the confined impinging jet technology when a few lead formulations have been identified. It is also crucial to consider the dead volumes in the mixers. In the CIJ, two jet mixers, the hold-up volumes are 50-100 microliters. This amount of material must be subtracted from the amount captured in the quench bath when calculating the recovery from the process. These losses are insignificant when operating on large scales but would account for 10% losses when total volumes of 5 mL are produced, as shown here. The impinging jet turbulent mixers are a valuable tool for producing LNPs at the GMP scale, as evidenced by the two FDA-approved COVID-19 vaccines.

Materials

Name	Company	Catalog Number	Comments
18:0 PC (DSPC)	Avanti Polar Lipids	850365P	Helper lipid
21 G x 1-1/2 in. BD PrecisionGlide Needle	BD	305167
96 Well Black Wall Black Bottom Plate	Fisher Scientific	07-000-135
96 Well White/Clear Bottom Plate, TC Surface	Thermo Fisher Scientific	165306
Acetic Acid, Glacial	Fisher Scientific	A38-212
ALC-0315	Avanti Polar Lipids	890900	Ionizable lipid
Amicon Ultra Centrifugal Filter, 100 kDa MWCO, 15 mL	Millipore Sigma	UFC910024
Amicon Ultra Centrifugal Filter, 100 kDa MWCO, 4 mL	Millipore Sigma	UFC810096
Bright-Glo Luciferase Assay System	Promega	E2620
Cholesterol	Millipore Sigma	C8667
CleanCap FLuc mRNA (5 moU)	Trilink Biotechnologies	L-7202
Confined Impinging Jets Mixer	Holland Applied Technologies, Helix Biotech, Diamond Tool and Die (DTD)	N/A	Contact Holland or DTD for custom orders and the Helix Biotech system is Nova BT. Review text for new mixer validation
D-Lin-MC3-DMA	MedChemExpress	HY-112251	Ionizable lipid
DMEM, high glucose, pyruvate	Thermo Fisher Scientific	11995065
DMG-PEG 2000	Avanti Polar Lipids	880151P	PEG-lipid
DODMA	Avanti Polar Lipids	890899P	Ionizable lipid
Ethanol 200 Proof	Decon Labs, Inc.	2701
Falcon 15 mL Conical Centrifuge Tubes	Fisher Scientific	14-959-70C
Falcon 50 mL High Clarity Conical Centrifuge Tubes	Fisher Scientific	14-959-49A
Fetal Bovine Serum, certified, United States	Thermo Fisher Scientific	16000044
HeLa	ATCC	CCL-2
HEPES, free acid	IBI Scientific	IB01130
HSW HENKE-JECT two-part 1 mL Luer	Henke Sass Wolf	4010.200V0
HSW HENKE-JECT two-part 5 mL (6 mL) Luer Lock	Henke Sass Wolf	4050.X00V0
Idex 1648 ETFE tubing ” OD 0.093” ID	Idex Health & Science	1648
Idex P-678 ¼”-28 to Luer fitting	Idex Health & Science	P-678
Idex P-940 ferrule for ETFE tubing	Idex Health & Science	P-940
Lipofectamine 3000 Transfection Reagent	Thermo Fisher Scientific	L3000001
Luer fitting	Idex Health & Science	P-604	Assemble on CIJ or MIVM mixer inlet with corresponding threads. Idex parts are also available through VWR and many other suppliers
Mixer stand	Holland Applied Technologies	N/A	See Markwalter &  Prud'homme for design.26 Contact Holland for Purchase
Multi-Inlet Vortex Mixer	Holland Applied Technologies and Diamond Tool and Die (DTD)	N/A	Contact Holland or DTD for custom orders. Review text for new mixer validation
O-ring (MIVM)	C.E. Conover	MM1.5 35.50 V75	Order bulk - consumable part. Ensure solvent compatibility if using an alternative source.
Outlet ferrule - CIJ	Idex Health & Science	P-200	Assemble with outlet fitting (large end flush with tubing)
Outlet fitting - CIJ	Idex Health & Science	P-205	Assemble with ferrule and tubing on CIJ chamber outlet
Outlet fitting - MIVM	Idex Health & Science	P-942	Combination with ferrule
Outlet tubing - CIJ	Idex Health & Science	1517	Use a tubing cutter for clean ends. Ensure extra tubing doesn't protrude into mixing chamber
Outlet tubing - MIVM	N/A	N/A	Fit to ferrule ID.
PBS - Phosphate-Buffered Saline (10x) pH 7.4, RNase-free	Thermo Fisher Scientific	AM9624
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
PHD 2000 Programmable Syringe Pump	Harvard Apparatus	N/A
Plastic two-piece syringe 1 mL	Thermo Fisher Scientific	S7510-1
Plug fitting	Idex Health & Science	P-309	Assemble on CIJ mixer sides (seal access point from drilling)
Quant-it RiboGreen RNA Assay Kit and RiboGreen RNA Reagent, RediPlate 96 RiboGreen RNA Quantitation Kit	Invitrogen by Thermo Fisher Scientific	R11491
Resazurin, Sodium Salt	Thermo Fisher Scientific	R12204
RNase AWAY Surface Decontaminant	Thermo Fisher Scientific	7000TS1
Scintillation vial	DWK Lifesciences	74504-20
SGE Gas Tight Syringes, Luer Lock, 100 mL	SGE	100MR-LL-GT
SGE Gas Tight Syringes, Luer Lock, 50 mL	SGE	50MR-LL-GT
Slide-A-Lyzer Dialysis Cassettes, 20 K MWCO	Thermo Fisher Scientific	66012
Sodium Acetate	Millipore Sigma	32319-500G-R
Sodium Hydroxide	Fisher Scientific	S320-500
Sucrose	Millipore Sigma	S7903-1KG
Syringe Filters, Sterile	Genesse Scientific	25-243
Triton X-100	Millipore Sigma	9036-19-5
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200056
Water, Endotoxin Free	Quality Biological	118-325-131	RNAse and DNAse free
Yeast RNA (10 mg/mL)	Thermo Fisher Scientific	AM7118