How is mRNA delivered to our cells?

Messenger Ribonucleic Acid (mRNA) requires a safe, effective and stable nano-delivery systems that protect from degradation as well as targeted cellular uptake and mRNA release¹. As discussed in the Particles 101 series, nanoparticles need to be designed by size, shape, charge and coating which can be optimised for mRNA cargo, targeted cellular uptake with negative charge and specific targeting coating and controlled release within the cells.

As we discussed previously in What Is mRNA?, mRNA can code for proteins and be used in cells as a translation step from DNA to RNA and transcription step from RNA to proteins. Taking this further we investigated Why Does mRNA work?, where we demonstrated the various tests knowing mRNA codes for a specific protein repeatedly and the protein functions correctly for the intended purpose. Also discussed is mass-production of mRNA which can be done using DNA source sequence, RNA polymerase and synthesised nucleotides to produce mRNA strands before degradation of DNA templates.

Why we need to protected mRNA

mRNA cannot be injected directly into the body and be expected to have a therapeutic effect. Similar to other drugs, mRNA needs to be optimised for the delivery mechanism (several routes available such as oral, injection and transdermal) and ensuring the correct location / cells. Without a delivery system, mRNA would be degraded by enzymes in the extracellular matrix and almost certainly would not enter mammalian cells.

Currently, 9 different RNA therapies have been delivered for therapeutic effects. Often RNA therapies focus on genetic manipulation affecting mRNA strands and the production of various proteins¹.

Almost always mRNA is delivered via injection directly to the site of application. This increases the bioavailability at the site of action compared to oral drug delivery routes where bioavailability is reduced due to the first-pass metabolism. It is important the injection site is easy to administer when encouraging mass adoption of a vaccine for example so it is not invasive which could deter patients.

How mRNA is protected

Currently, the most common method of encapsulating mRNA is using liposome nanoparticles². Liposomes are similar to the cells within our body and have been approved for FDA approval for many different drugs on the market. Currently, there are over 30 liposomal drugs approved on the market encapsulating RNA, molecules and proteins for therapeutic effect³.

mRNA is encapsulated likely using microfluidic droplet formation where mRNA can be mixed with liposomes within the discrete phase where the continuous phase self-assembles the liposome nanoparticle with mRNA contents⁴. This can be created in a very accurate and continuous manner however there are some questions over the solvents used, the microfluidic chip designs and lipid concentration⁵. A recent article regarding mRNA COVID vaccines stated “erotic mixing technology” which likely builds upon the microfluidic droplet formation devices in the literature⁶. More about simulating droplet microfluidics is available here. Other forms of manufacture such as the DELOS method have a mixed solution of lipids and mRNA which produce liposomes by dissolving in an organic solvent and under carbon dioxide pressure⁷.

RNA is loaded into liposomes by the electrostatic association between the negatively charged RNA strands and positively charged lipid heads when forming liposomes⁸. Liposome formation often self-assembles due to the non-polar lipid tails which can form micelles and liposomes with positively charged inner core and outer surface. The electrostatic association means RNA can be attached to the inside and outside of liposome nanoparticles where the microfluidic manufacture needs to be optimised for internal loading. Additionally, the construction of the different lipids can affect the overall liposome charge as well as adding additional surface coatings⁹. This is similar to the discussion about the importance of charge and surface coatings which can dramatically affect the nanoparticle distribution, cellular uptake and internalisation. As mentioned in a recent article, “proprietary, bespoke lipids” are used for the mRNA COVID vaccines⁶. One common surface coating which may have been used is a lipid-PEG (Poly(ethylene glycol)) which helps the liposomes evade the immune system and optimise delivery to the target cells¹⁰.

Conclusion

Nanotechnology, nanoparticles in particular, allow for the delivery of mRNA to cells to produce proteins. Without nanoparticles, it would be very difficult to deliver mRNA to cells. Without liposome nano-carriers, mRNA would be degraded before having any therapeutic effect. Whilst mRNA delivery for vaccines is novel, the process has existed for several years using mRNA for other therapies. Similarly, liposomes have been used for numerous therapeutic applications. Overall, it is exciting to see the developments using nanotechnology for mRNA vaccines.

References

1. Kim, Y.-K. (2020). RNA Therapy: Current Status and Future Potential. Chonnam Medical Journal, 56(2), 87. https://doi.org/10.4068/cmj.2020.56.2.87
2. Hou, X., Zaks, T., Langer, R., & Dong, Y. (2021). Lipid nanoparticles for mRNA delivery. Nature Reviews Materials, pp. 1–17. https://doi.org/10.1038/s41578-021-00358-0
3. Beltrán-Gracia, E., López-Camacho, A., Higuera-Ciapara, I., Velázquez-Fernández, J. B., & Vallejo-Cardona, A. A. (2019). Nanomedicine review: Clinical developments in liposomal applications. Cancer Nanotechnology, Vol. 10, pp. 1–40. https://doi.org/10.1186/s12645-019-0055-y
4. Kawamura, J., Kitamura, H., Otake, Y., Fuse, S., & Nakamura, H. (2020). Size-Controllable and Scalable Production of Liposomes Using a V-Shaped Mixer Micro-Flow Reactor. Organic Process Research and Development, 24(10), 2122–2127. https://doi.org/10.1021/acs.oprd.0c00174
5. Carugo, D., Bottaro, E., Owen, J., Stride, E., & Nastruzzi, C. (2016). Liposome production by microfluidics: Potential and limiting factors. Scientific Reports, 6(1), 1–15. https://doi.org/10.1038/srep25876
6. King, A. (2021). Why manufacturing Covid vaccines at scale is hard. Chemistry World – Royal Society of Chemistry website: https://www.chemistryworld.com/news/why-manufacturing-covid-vaccines-at-scale-is-hard/4013429.article
7. Maja, L., Željko, K., & Mateja, P. (2020). Sustainable technologies for liposome preparation. Journal of Supercritical Fluids, Vol. 165, p. 104984. https://doi.org/10.1016/j.supflu.2020.104984
8. Sayour, E. J., Mendez-Gomez, H. R., & Mitchell, D. A. (2018). Cancer vaccine immunotherapy with RNA-loaded liposomes. International Journal of Molecular Sciences, Vol. 19. https://doi.org/10.3390/ijms19102890
9. Kang, E. C., Akiyoshi, K., & Sunamoto, J. (1997). Surface coating of liposomes with hydrophobized polysaccharides. Journal of Bioactive and Compatible Polymers, 12(1), 14–26. https://doi.org/10.1177/088391159701200102
10. Kim, J., Eygeris, Y., Gupta, M., & Sahay, G. (2021). Self-assembled mRNA vaccines. Advanced Drug Delivery Reviews, Vol. 170, pp. 83–112. https://doi.org/10.1016/j.addr.2020.12.014