optimized formulation of lipid nanoparticles using a two-jet continuous impinging jet (cij) mixer

Syntetisering av lipidnanopartiklar med hjälp av CIJ- och μMIVM Mixer-tekniker

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 &#34;endosome escape&#34; 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 conditions12,13,16. 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 &#34;fouling&#34; of the mixing device, and there are problems with uniformity and stability of flows that make microfluidics scaleup challenging for industrial-scale production18,19. 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 mechanism21,22,23. 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, &#964;mix, and the nuclei growth period, &#964;agg. The dimensionless Damk&#246;hler number, defined as Da = &#964;mix/&#964;agg, captures the interplay between these time scales24. In instances of slow mixing (Da &#62; 1), the final size of LNPs is transport-controlled and varies with mixing time. Conversely, during fast mixing (Da &#60; 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 &#964;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 &#34;confined impinging jet&#34; (CIJ) mixer. Turbulent vortices produce solvent/antisolvent striation length scales of 1 &#181;m 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 = &#931; (ViDi/vi), where Vi is the flow velocity in each steam, vi is the kinematic viscosity of each stream, and Di 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 Damk&#246;hler number. Another type of mixer is the &#34;multiple inlet vortex mixer&#34; (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.

Författare i fokus: Förbättra bildandet av lipidnanopartiklar genom turbulent blandning i begränsade geometrier

Syntetisering av lipidnanopartiklar genom turbulent flöde i slutna impinging jet-blandare

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 to 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.

Optimerad formulering av lipidnanopartiklar med hjälp av en tvåstrålig CIJ-mixer (Continuous Impinging Jet)

optimized-formulation-lipid-nanoparticles-using-two-jet-continuous

Research

JoVE Journal

Bioengineering

Optimized Formulation of Lipid Nanoparticles Using a Two-Jet Continuous Impinging Jet (CIJ) Mixer

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

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.

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 (&#181;MIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of ...

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.

Advanced Formulation and Production of Lipid Nanoparticles Using a Four-Jet Micro-Size Multiple Inlet Vortex Mixer (&#181;MIVM)

The Multiple Inlet Vortex Mixer has tangentially injected streams into the mixing chamber that form lamely in the rotating vortex. A high Reynolds number in the mixing chamber ensures the production of micron scale turbulent vortices, which rapidly mix the lamely, 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 the 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 antis solvent 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 lure 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 four 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.