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.
