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Microfluidics to power better LNP-mRNA preparation

Bowen Tian, Senior Application Scientist,  Particle WorksBowen Tian, Senior Application Scientist, Particle Works
Lipid nanoparticles (LNPs) have been successfully used for the delivery of mRNA, including the billions of doses of the Moderna and Pfizer-BioNTech COVID-19 vaccines that have been administered globally. The unprecedented success of LNP-mRNA nanoparticles has led to a focus on the development of novel genomic medicines to meet clinically unmet needs, such as ‘cures’ for tumors and genetic diseases.

LNPs are based on well-established liposome technology, with the addition of ionizable or cationic lipids for encapsulation with negatively charged oligonucleotides – such as RNA and DNA –through electrostatic interactions. Liposomes were first discovered by British scientist Alec D Bangham in the 1960s. This discovery was followed by several generations of hard work by liposome scientists, which resulted in FDA approval of the first liposomal drug, Doxil® – a liposomal preparation of the chemotherapy drug doxorubicin that greatly reduces the cardiotoxicity associated with the drug itself – in 1995. Many of the leading scientists currently working on LNPs were involved in this early liposomal research, including Professor Pieter Cullis, who has driven the field forward over the past 40 years. His team developed ionizable lipids that helped the LNP-mRNA vaccines to reach clinical trials within a three-month timeframe during the COVID-19 pandemic.

It is widely known that COVID-19 vaccines – such as those from Moderna and Pfizer-BioNTech – use LNPs to deliver mRNA into cells, where it is expected to be released to produce proteins of therapeutic interest. However, recent studies show that less than four percent of LNP-mRNA nanoparticles are capable of efficiently releasing mRNA intracellularly.1-4 This means that the majority of the costly LNP-mRNA nanoparticles administered are not effective therapeutically, rendering the overall process highly inefficient.

This invites the question ‘Why does this happen?’. One of the key reasons lies in the preparation and manufacture of LNP-mRNA nanoparticles. The traditionally-used batch methods cannot produce homogeneous nanoparticles in terms of size, morphology, lipid composition or the amount of mRNA payload. This leads to significant variation in the behaviors of LNP-mRNA nanoparticles in vivo. Another contributory factor may be low endosomal escape, which is not yet fully understood.

Currently, significant efforts are being made to explore novel methods for the preparation and manufacture of LNP-mRNA nanoparticles. The ultimate goal is to produce particles with identical lipid compositions – for example, ionizable lipids, helper lipids, cholesterol and PEG-lipids – and the same amount of mRNA payload per individual nanoparticle. Although this is very challenging, it would provide significant clinical benefits and have a positive impact on novel genomic medicine development using LNPs.

Why it is so difficult to make LNP-mRNA nanoparticles?

LNP-mRNA nanoparticles are formed through a self-assembling process that is very difficult to control. Many efforts have been made to improve LNP-mRNA nanoparticle formation through precision mixing between lipids and mRNA. An example of this is a widely used ethanol injection method, where a syringe is used to inject lipids suspended in ethanol drop-by-drop into aqueous mRNA solutions. T-mixing is another option, and is mostly used in large-scale production. However, both methods fail to produce truly monodisperse LNP-mRNA nanoparticles, and lack batch-to-batch consistency.
Another challenge to overcome is the shear force, which could accelerate the degradation and rupture of these LNP-mRNA nanoparticles. Protection of mRNA is important for several reasons. These include the avoidance of early enzymatic degradation – especially in vivo – prevention of overstimulation of the immune system and, crucially, for endosomal escape to release mRNA inside of cells. The LNP structure also affects the result, so high shear forces should be avoided. These requirements make microfluidics an attractive option for LNP-mRNA nanoparticle preparation.

Microfluidic devices provide unique advantages compared to ethanol injection and T-mixing, such as:

• Precision mixing of different fluids at microliter volumes – typically from one to a few microliters of active mixing volume within the microfluidic chip.

• Lipids in ethanol and mRNA in water can be continuously flowed into the chip from two separate channels, meeting at the junction to form LNP-mRNA nanoparticles.

• An optimized microfluidic set-up enables production of highly monodisperse LNP-mRNA nanoparticles, which are expected to have improved homogeneity across the population in terms of lipid composition, mRNA payload and morphology.

Microfluidic workflows with carefully optimized lipid formulations, flow rates and mRNA cargoes have the potential to produce homogenous LNP-mRNA nanoparticles significantly superior to alternative manufacturing methods. They offer better control of particle production, making it easier to select the formulation that works best in terms of both maximized therapeutic effects and minimized side effects. This would potentially allow the injection of mRNA vaccines at a significantly reduced dose, reducing the risk for vulnerable people who require more frequent boosters. It may also create a new therapeutic window that allows a much higher dose of mRNA to be effectively delivered, to maximize the therapeutic effect. More importantly, this could greatly progress genomic medicine, offering a solution to currently unmet clinical needs.

At Particle Works, we are building on over 20 years of engineering experience in microfluidics systems to release game-changing particle generation platforms that allow our customers to unlock the true power of particles with microfluidic technology. We are working to provide automated solutions for LNP-mRNA nanoparticle engineering, and offer platforms that support our customers through their entire particle development journey – from screening and discovery, through preclinical development and process optimization to large-scale GMP manufacture – with an aim to make every single nanoparticle perfect.

Microfluidic devices provide unique advantages compared to ethanol injection and T-mixing.

For more information on our automated particle generation platforms, visit

1. Maugeri, M., Nawaz, M., Papadimitriou, A. et al. Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells. Nat Commun 10, 4333 (2019).

2. Munson, M.J., O’Driscoll, G., Silva, A.M. et al. A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery. Commun Biol 4, 211 (2021).

3. Dowdy, S. Overcoming cellular barriers for RNA therapeutics. Nat Biotechnol 35, 222–229 (2017).

4. Kaczmarek, J.C., Kowalski, P.S., Anderson, D.G. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med 9, 60 (2017).
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