The remarkable success of mRNA Covid-19 vaccines was far from guaranteed until scientists managed to optimise a 30-year-old technology and turn lipid nanoparticles into high-performing delivery units. By Manuela Callari.

Vaccines and lipid nanoparticles

Professor Pall Thordarson from UNSW Sydney.
Professor Pall Thordarson from UNSW Sydney.
Credit: Supplied

By now millions of people around the world have received a Pfizer-BioNTech or Moderna Covid-19 vaccine. These shots use mRNA, a single-stranded sequence of nucleotides containing the instructions to produce the spike protein SARS-CoV-2 uses to enter our cells.

When delivered into a cell, the mRNA (messenger ribonucleic acid) enters protein factories called ribosomes that read the code and produce the spike protein. The spike is recognised as a dangerous invader by our immune system, which then starts producing antibodies that can protect us when we come into contact with the real invader.

But mRNA is incredibly unstable, says Archa Fox, one of Australia’s leading experts in RNA biology and an associate professor at the University of Western Australia. It is susceptible to temperature, which is why mRNA Covid-19 vaccines need to be stored at very cold temperatures. It is also highly vulnerable to enzymes in our body that chop it into pieces, making delivering mRNA to cells a daunting task.

“Without having a coating, the RNA injected intramuscularly would just break up before it even reaches our cells,” says Fox.

In addition to these sensitivities, mRNA strands are large, negatively charged molecules and can’t cross the cell membrane.

With such seemingly insurmountable challenges, many scientists and tech ventures were doubtful the technology could ever work. But then a solution was found in a technology older than the idea of mRNA therapeutics itself. The fragile mRNA molecules were packed into tiny balls of fat called lipid nanoparticles (LNPs).

Lipids are long molecules with a polar, water-soluble head and a nonpolar, hydrophobic tail. When put in water, lipid molecules aggregate into tiny spheres so that all the tails are close to each other in the core of the sphere and away from the water, while the water-liking polar heads make up the sphere’s surface.

The LNPs used in the Covid-19 vaccines contain four ingredients: ionisable lipids that bind to the negatively charged mRNA; pegylated lipids that have a polyethylene glycol (PEG) strand attached to the lipid head and improve water solubility; neutral lipids; and cholesterol molecules that contribute to the particle’s structure.

The nanoparticle protects mRNA from thermal degradation and shields it from destructive enzymes while shuttling the messenger to the cell. The cell swallows the LNP through receptor-mediated endocytosis – a natural process cells use to absorb metabolites, hormones, proteins and even viruses. When the LNP binds to the cell’s membrane, a larger lipid bubble called an endosome wraps the LNP and shunts it into the cell. The endosome’s acidic interior triggers the dissociation of the LNP, which releases its mRNA cargo into the cell’s cytoplasm. Once released, the mRNA is free to do its job.

The concept seems simple, but perfecting it was far from straightforward.

Over more than three decades, researchers have designed and tested hundreds of promising LNPs, yet only a handful of these systems have reached the clinic. Scientists had to work hard to overcome the many pitfalls nanoparticles present.

When engineering LNP for vaccine delivery, researchers must formulate a lipid sphere that is stable enough to give the vaccine a long shelf life. Each batch must contain particles of the same size and charge, and there should be no differences from batch to batch.

The manufacturing process needs to be easily scalable with costs kept low. “You want vaccines to be as cheap as possible because you need to produce enough for everyone,” says Dr Mariusz Skwarczynski, a research fellow at the University of Queensland who studies nanoparticles for the delivery of drugs and vaccines.

Once a promising LNP is engineered in the lab, it must be tested in preclinical studies. Positively charged lipids are inherently toxic, says Skwarczynski. “First of all, you need to check that the particles are safe,” he says. “There are delivery systems for RNA that are very efficient, but we can’t use them in humans because of their toxicity.”

Often, researchers find very little correlation between in vitro studies and animal studies. What works well in a petri dish isn’t always replicated in vivo.

One hurdle involves eluding the body’s immune response to LNPs. “The immune system is always on the lookout for foreign identities,” says Professor Pall Thordarson, a nanoparticles expert from UNSW Sydney. “Nanoparticles elicit an immune response because they pretty much look like a virus to the body.”

The last, not-less-trivial step is to create an LNP that can fall apart once it reaches its target. “Releasing [the cargo] into the cell has always been the hardest job,” says Thordarson.

Overcoming these stumbling blocks has taken decades of hard work and billions of dollars of investment. When BioNTech and Moderna bet on mRNA therapeutics and vaccines about 10 years ago, solving the delivery problems was a top priority. BioNTech and Moderna engineers have tweaked the different parameters, such as the lipid formulation or the particle formation methods, until they created the high-performing LNPs now used in Covid-19 vaccines.

“[These kinds] of nanoparticles have been known for almost 30 years, but they are considered somewhat primitive,” says Thordarson. “These companies have managed to optimise their lipid nanoparticle systems to work so well as they do. I really take my hat off to them. It is seriously impressive.”

BioNTech and Moderna are now working on updating their Covid-19 vaccines to include new variants. By slightly modifying the mRNA code to match the genetic code of the new strains of the virus, they can reuse the same LNP formulation. Now that they know their LNPs are safe and work well, they can use them for different vaccines.

But the research work on LNPs has not stopped. Optimising the nanoparticles even further could lead to vaccines that require lower doses, which could ease the manufacturing burden amid a pandemic.

Developing new LNPs will likely take too long to make a difference during this pandemic, but biotech companies are continuing to look for better ways to get mRNA into cells for various applications.

Scientists have long known the potential of LNPs in medicine, but the Covid-19 pandemic has reinvigorated interest in these delivery systems. The success of LNPs in mRNA vaccines is exciting because LNPs can be applied to a huge array of drugs that need to be delivered inside cells.

“The most obvious one is cancer,” says Professor Martina Stenzel from UNSW Sydney, who studies nanoparticles for the delivery of chemotherapeutics. “The use of nanoparticles in cancer by far outweighs everything else. People always felt that the nanoparticles are really getting lodged in tumours, and that was first observed in animal studies in the ’90s.”

Nanoparticles can improve anticancer drugs’ solubility, stabilise them and enhance their accumulation in tumours, she explains. In theory that means patients can receive a lower dose of drugs, reducing side effects.

“We have never seen a really massive improvement to justify very expensive clinical trials,” says Stenzel. “But RNA-based drugs is where nanoparticles can really make a difference because these are cleared too quickly from the bloodstream.”

Stenzel says one of the main challenges in the field has been a lack of collaboration between material scientists and clinicians and the difficulty securing funding for multidisciplinary projects because each funding body narrowly looks at its own field.

“What we need is more of a concerted effort where these two groups work together more closely,” she says. “I’m personally very excited to see that there is finally a product that combines mRNA and lipid nanoparticles. It’s just a bit sad that we needed a pandemic for people to wake up and see the importance of medical and fundamental research.”

This article was first published in the print edition of The Saturday Paper on July 31, 2021 as "Lipid service".

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Manuela Callari is a freelance science writer.

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