The Role of Lipid Nanoparticles in Vaccine Development
The recent approval of COVID vaccines from Moderna and Pfizer based on mRNA-containing lipid nanoparticles (LNPs) has propelled this pioneering technology, shifting it from being simply viewed as speculative research to becoming transformative in the area of genetic medicines. The pharmaceutical world has seen vaccine development experience a sharp jolt, evolving from the 1950’s concept of one egg, one vaccine dose and the bulk cellular expansion in bioreactors, to a highly efficient and timely manufacturing protocol. The advent of mRNA containing LNPs has enabled a highly effective new vaccine platform, but at the same time has raised many questions.
Why has this new technology changed our dependence on cell cultured vaccines? Will the NanoAssemblr democratise vaccine production globally?
The 4 pillars of RNA vaccine development
Classically there are four pillars of vaccine development, individually they are inconsequential; but together they can be a formidable assault on pathogenic viral invaders! These pillars are Antigens, Vectors, Delivery-Nanoparticles, and Manufacturing. Each pillar has its set of design and development considerations and associated technologies.
To streamline understanding of each pillar it is important to explore these further details; an Antigen is what the body defences identify as foreign and will attack. In the case of COVID much of the focus has been on the characteristic spike protein. Some of the vaccines are designed to create a copy of this passive component of the virus to educate the immune system to respond by producing antibodies. A viral vector is generally a harmless virus (often an Adenovirus) that is hijacked to carry the genetic code of interest (ie the spike protein) into the cells, like a letter is placed into an envelope to be posted. In this discussion consider the Vector as a replicon virus such as an Alphavirus where the structural protein code is substituted for the Gene Of Interest (GOI), being the spike protein, but retaining the four non-specific proteins (NSP) that work together to actively replicate the GOI once in the ribosome – ultimately producing a huge amount of antigens, a self-amplified RNA 1. A bit of RNA code is quite vulnerable, your body is trained to destroy free floating RNA, so it needs to catch an uber to enter the body and ultimately the cells. This uber needs to be able to navigate around our defences. To do this, a suitable camouflage is needed.
A Lipid Nanoparticle (LNP) is perfect to encapsulate the code thereby becoming the delivery pillar and it has huge scalable potential for manufacturing, which is the next pillar. Whilst it is a challenge to bring all these components together, the ultimate challenge is to manufacture to scale. Translating research to usable medicine is often a bottleneck and many candidates fall over at this stage. It is exceptionally important to de-risk the process well before this stage. This is where the Precision Nanosystems platform products – the NanoAssemblr range – have had a huge impact in translating research to the clinic.
The anatomy of lipid nanoparticles (LNP)
Often the terms Liposome and LNP are used interchangeably, however, whilst they are similar in many ways, there are distinct differences in their function and structure. Consider a Liposome is made up of a Lipid bilayer primarily composed of amphipathic phospholipid enclosing an interior aqueous space, it can be decorated with a protein adding targeted delivery to its capabilities. LNPs can take on a variety of forms enhancing their ability to encapsulate an assortment of cargoes like peptides, genetic payloads like siRNA, mRNA and saRNA plus other small molecules.
The most exciting of these are those formulated with ionisable cationic lipids. Recently research from Meng and Grimm proposes LNPs composed of the best-performing iPhos and different helper lipids—zwitterionic lipids, ionizable cationic lipids and permanently cationic lipids—achieved selective organ targeting (SORT) and organ-specific CRISPR-Cas9 gene editing in spleen, liver, and lungs of mice, respectively3 .
Mechanisms of LNP action and the role of different lipid components
The LNP structure paves the way for a nanoprecipitation method for their creation. Precision Nanosystems employ a microfluidic architecture that allows two streams to mix under laminar flow conditions enabling the process to be reproducible and scalable. Importantly this mixing rate is rapid, <1 millisecond adhering to the tenent ‘the mixing rate should be faster than assembly rate’.
Consider one of these streams is aqueous, a buffer, such as PBS to maintain the pH whilst carrying the payload. The other stream has the lipid components dissolved in a solvent such as ethanol. Controlling the flow rate ratios and the total flow rate will vary the nanoparticle size. A LNP is not a solid sealed system like a rubber ball, instead it is a collection of elements that are bound by charge. In a mRNA example, the cationic lipids meet the negative charges of the phosphate backbone of the mRNA. Then the neutral helper lipids such as zwitterionic lipids stabilise the lipid bilayer of the LNP. This enhances delivery efficiency, and thanks to the aqueous environment, polyethylene glycol (PEG)-lipid forms the outer shell pointing it’s hydrophobic tails inwards. These improve the colloidal stability in biological environments by reducing specific absorption of plasma proteins and forming a hydration layer over the nanoparticles4. The result is unilamellar, predictably sized, uniform LNPs created in a matter of seconds.
Precision Nanosystems’ NanoAssemblr series facilitates the encapsulation of genetic material into ionisable cationic lipids ideal to be seen as ‘self’ by the body – moving by stealth into the cells by endocytosis. The cellular uptake of LNP mainly relies on the endocytic pathway. More in detail, it has been shown that specific serum proteins adsorbed on the surface of LNPs upon intravenous injection can drive the cell internalisation5. For an effective nucleic acid delivery, a large portion of functional molecules should escape the endosomal compartment before the degradation cascade begins. Ionizable lipids, which are capable of modulating their charge depending on the environmental pH, are recognised as a key component of LNPs for the endosomal escape6.
NanoAssemblr for vaccine development
Lipid nanoparticles (LNPs) are the most clinically advanced non-viral gene delivery system. Lipid nanoparticles safely and effectively deliver nucleic acids, overcoming a major barrier preventing the development and use of genetic medicines and vaccines. The Precision Nanosystems platform facilitates the formulation of LNP vaccines on a research scale through to full GMP manufacture. The nanoparticles produced work better than other methods of manufacture such as T-Tube mixing, in a study siRNA-LNPs manufactured by three NanoAssemblr® instruments exhibited encapsulation efficiencies of higher than 95%, Factor VII siRNA knockdown efficacy was maintained for nanoparticles produced on the NanoAssemblr® Benchtop, Blaze, and GMP System.
Particles generated by the NanoAssemblr® platform are more uniform than those made by conventional T-Tube mixing methods. T-tube generated lipid nanoparticles exhibit a multilamellar morphology vs the homogeneous-core structure for the NanoAssemblr® generated lipid nanoparticles. Serum Factor VII siRNA knockdown efficacy was higher for NanoAssemblr® siRNA lipid nanoparticles compared to conventional T-tube lipid nanoparticles, 72 hours following systemic administration7.
Your nano solution
ATA Scientific are just one call away to demonstrate the rapid, effortless lipid nanoparticle production, and optimisation. We can tailor the system choice to suit your stage of development.
ATA Scientific have a portfolio of products that complement the NanoAssemblr range such as the Malvern Panalytical Zetasizer, ITC and Nanosight plus microscopy and cell counting solutions. Contact us today for an illuminating discussion.
1) Self-Amplified RNA Vaccine Against COVID-19. https://youtu.be/tVh1s06H_nw
2) Liposomes vs. Lipid Nanoparticles: Which Is Best for Drug Delivery? https://www.biochempeg.com/article/122.html Accessed 24 Sept 2021
3) Meng, N., Grimm, D. Membrane-destabilizing ionizable phospholipids: Novel components for organ-selective mRNA delivery and CRISPR–Cas gene editing. Sig Transduct Target Ther 6, 206 (2021). https://doi.org/10.1038/s41392-021-00642-z
4) Cullis, P. R., and Hope, M. J. (2017). Lipid nanoparticle systems for enabling gene therapies. Mol. Ther. 25, 1467–1475. doi: 10.1016/j.ymthe.2017.03.013
5) Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 2020;15:313-320. https://doi.org/10.1038/s41565-020-0669-6.
6) Schlich, M, Palomba, R, Costabile, G, et al. Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles. Bioeng Transl Med. 2021; 6:e10213. https://doi.org/10.1002/btm2.102137) https://www.precisionnanosystems.com/workflows/formulations/lipid-nanoparticles