How mature is LNP technology?

We've barely scratched the surface

Nov 01, 2022

The COVID-19 mRNA vaccines use lipid nanoparticles (LNPs) as “carrier vehicles” for the mRNA. These LNPs are balls of fat that serve to protect the mRNA and get them into cells.

How much do we understand about how safe LNPs are, or how they even work? How mature is LNP technology?

That’s what we’ll cover in this article.

“The future is going to be not in lipid nanoparticles”

It’s surprising to read what some researchers were saying about LNPs just a few years before the COVID-19 vaccine rollout. Let’s look at this article from March 2017: Worth the RISC?

It talks about the use of LNPs in delivering RNAi therapeutics. RNAi therapeutics work by delivering certain types of RNA (different from mRNA) into cells, which will induce the cells to silence specific genes.1

Lipids, although still in use… have, as a class, never overcome toxicity concerns stemming from innate immune activation, especially with chronic use.

They mention Alnylam, an RNAi therapeutics company that has done extensive research into developing LNPs. In fact, Alnylam claims that their LNP technology is a component of the COVID-19 vaccines and is seeking royalties from Moderna and Pfizer (more here and here).

Anyway, here was what was being said in 2017 about one of their LNP-encased drugs:

Alnylam's LNP-delivered composite… showed dose-dependent cytokine and complement2 activation in phase 1, with related side effects, even though patients were given steroid premedication. The drug, outlicensed, has not returned to the clinic.

In other words, this lipid-encapsulated product was too inflammatory even when given with steroids, which are anti-inflammatory drugs.

Alnylam did eventually get an LNP-encased drug, patisiran (otherwise known as Onpattro), FDA-approved, though it needed to be administered with steroids:

Patisiran uses a different cationic lipid and has not shown the same increase in inflammatory markers, but it still must be given with steroids.

The article quotes from Dirk Haussecker,3 former RNAi researcher and founder of the RNAi Therapeutics blog (emphasis mine):

But for siRNA delivery, LNPs "are out of fashion," says Haussecker. "There's a lot of clinical data now that show that especially for chronic dosing that you shouldn't be using them."

“siRNA” stands for small interfering RNA, which is a type of RNA that gets used in RNAi therapeutics.

Judy Lieberman, a researcher at Harvard and member of Alnylam's scientific advisory board, seemed to concur and even said:

"The future is going to be not in lipid nanoparticles," says Lieberman.

Given that we’ve just rolled out LNP-encased COVID-19 vaccines all across the globe, I guess she was wrong.

But different LNPs have different safety profiles, and surely the ones in the COVID-19 mRNA vaccines aren’t as inflammatory, right? Surely we’ve solved the issue, and now we have LNPs that are fine for “chronic dosing,” like yearly booster vaccines?

They’re highly inflammatory

Unfortunately there’s no evidence that the problem was solved.

In fact, this very recent paper from March 2022 showed that LNPs containing the same lipid (SM-102) that was used in the Moderna COVID-19 vaccine, induced high systemic levels of inflammation. The LNP alone, aka empty LNPs without any RNA load, was sufficient to cause potent release of inflammatory cytokines.4

This is in line with other studies showing that LNPs can be highly inflammatory, like this study from December 2021, which showed that the LNPs used in various RNA products undergoing human clinical trials5 led to “rapid and robust inflammatory responses, characterized by massive neutrophil6 infiltration... and production of various inflammatory cytokines” when injected intradermally or intramuscularly in mice.

The same dose delivered intranasally led to massive inflammation in the lungs and a high mortality rate in mice.

Why are these LNPs so inflammatory or toxic?

According to University of British Columbia nanoparticle scientist Pieter Cullis, it has to do with the fact that they contain cationic, or positively charged lipids:

“There are no cationic lipids in nature,” Cullis says. “And we knew we couldn’t use permanently positively charged lipids because they are so damn toxic.” Those lipids would rip cell membranes apart, he adds.

Components of LNPs. *PEG* = polyethylene glycol. Image from here.

Now, the LNPs that we’ve been using for the mRNA vaccines use ionizable lipids which are supposed to be charged only under certain conditions. These are supposed to be safer than lipids that are permanently positively charged.

Then why are they still so inflammatory?

What do we know about how LNPs work?

The truth is, we don’t have a good grasp of how LNPs work.

Here’s how we think they work: The LNPs are normally neutrally charged. They are supposed to get taken up by cells via a process called “endocytosis,” whereby they become encapsulated within organelles (substructures within cells) called endosomes, which are membrane-bound sacs. The inside of endosomes are acidic. Only once the LNPs are within the acidic endosomes, do they become positively charged.

Once the LNPs are positively charged, we think this triggers a change in the shape of the LNPs, which helps the LNPs break free from inside the endosomes and ultimately release its RNA cargo into the cell’s cytoplasm, where the RNA is free to do its job.

Image from here, showing how the IVT (in vitro transcribed) mRNA encased in LNP gets taken up by the cell.

This is what we think happens, but we’re not exactly sure.

From a very recent paper from Feb 2022:

The precise sites and mechanisms whereby LNPs help mRNA to escape from the endosomal lumen are, to date, mysterious.

The “endosomal lumen” just means the interior of the endosome. So we don’t understand how the mRNA eventually escapes the inside of endosomes.

Even when cells take up the LNPs, it doesn’t guarantee that the RNA will escape from the endosomes.

Below are results from experiments where they took mRNA encoding for GFP, which is a fluorescent protein, and encased it in 6 different kinds of LNPs. It shows that more uptake of LNP did not always correlate with more GFP expression.

In figure A you’ll see that cells that got the mRNA encased in L608 LNP expressed the most GFP. However, according to figure B, L608 did not have the highest uptake by cells. The MC3 and ACU5 LNPs had higher uptake, but did not have GFP expression that was as high.

So the amount of LNP uptake or internalization is not the only factor to predict successful expression of mRNA. This suggests that some of the LNP that’s taken up by cells gets trapped within endosomes.

Are LNPs getting trapped, causing cytotoxicity?

Indeed, often the LNPs don’t successfully escape endosomes, and this causes all kinds of issues:

For some LNP-mRNA formulations, a large fraction accumulates in a small population of early endosomes that are defective in new cargo uptake and arrested in maturation.

Endosomes come in different types, and “early endosomes” are a type of endosome. They undergo changes to “mature” into “late endosomes.”

Image taken from here showing the transition from early to late endosomes. Rabex5, Rab5, Rab7, etc. are proteins associated with different types of endosomes.

So it seems that sometimes the LNPs accumulate in early endosomes in a “maturation-arrested state”:

a significant fraction of endosomal structures with accumulated LNPs is in a maturation-arrested state and insulated from normal cargo uptake.

As early endosomes mature into late endosomes, they become increasingly acidic. However, LNPs can mess with this maturation process:

LNP accumulation is accompanied by endosome acidification defects.

In fact, it seems the ionizable lipids of LNPs may interfere with V-ATPase, which is an enzyme that maintains pH in endosomes:

ionizable LNP lipids may interfere with V-ATPase activity or increase the leakiness of endosomal membrane to protons.

One of the LNPs they tested was similar to the LNP used in the Moderna mRNA vax, and this was one of the ones that messed with endosome acidification:

Interestingly, MOD5 (an analogue of the lipid used in mRNA-1273 SARS-CoV-2 vaccine) also had an impact on acidification, albeit less than MC3.

This may lead to some of the cytotoxic effects that we see with LNPs:

In addition, acidification is so critical to various endosomal activities, such as protein sorting, endosomal progression, lysosomal degradation, and cellular homeostasis, that if compromised, will lead to a series of cytotoxic consequences.

And:

In addition to cytotoxicity, these alterations may cause an inflammatory response similar to the immune system defects characterizing lysosomal storage disorders. Therefore, defective endosomal acidification may account for a great deal of the cytotoxic effects of LNPs.

There are numerous ways in which LNPs could be toxic:

The reasons for cytotoxicity are diverse, comprising oxidative stress and apoptosis.

And by the way, suppressed acidification may prevent biodegradation of the LNPs:

As endolysosomal hydrolytic enzymes require acidic pH, suppressed acidification will prevent the biodegradation of the LNP lipids (per se biodegradable), thus exacerbating LNP accumulation further.

So to sum up:

LNPs get taken up by endosomes in cells. In order for the LNPs to ultimately get released by the endosomes, the endosomes need to be acidic. However, the LNPs may interfere with the process that maintains acidity within endosomes. This would cause some LNPs to remain stuck in the cells, which would lead to cytotoxic effects, and prevent biodegradation of the LNPs.

Does this partially explain why we see the vax mRNA lasting for months in some people?

What happens to the mRNA inside LNPs that are stuck within endosomes? Could those arrested endosomes actually serve as long-term reservoirs for the mRNA? After all, we now know that the vaccine mRNA can survive for months in the body.

Or perhaps the stuck mRNA eventually gets moved to some other reservoir within the cell?

mRNAs that are not being used can sometimes aggregate in compartments in the cytoplasm called p-bodies or stress granules; sometimes these are sites of mRNA degradation, but sometimes they act as storage compartments. Both p-bodies and stress granules are poorly understood. How do they interact with endosomes?

Endosomes, by the way, are more than just membrane-bound sacs.

Furthermore, the complexity of the endosomal network cannot be underestimated. It consists of populations of organelles that are subcompartmentalized, dynamically exchange cargo and trafficking machinery, and change in size and position over time…

Do we know if stuck mRNA, whether in arrested endosomes or elsewhere, can’t eventually be released and translated? Perhaps a change in metabolic state of the cell, or some other change, can release that mRNA?

If so, could this lead to people having an adverse reaction to the vaccine weeks or even months post-vaccination?

The LNPs end up all over the place

Here’s another undesirable aspect of the LNPs: we don’t have good control over where they end up.

The COVID-19 mRNA vaccines are injected intramuscularly, and we were told that most of it would stay close to the injection site. But we now know that’s not true. For example, if you look at the last figure in the supplementary material of this study, you’ll see that mice injected with the Pfizer vaccine had mRNA in heart tissue, even when injected intramuscularly:

“RBD” stands for “receptor binding domain.” This shows COVID-19 mRNA spike RBD gene copies relative to expression of the β-actin gene, which is commonly used as a way to normalize gene expression because its expression is thought to not change much with different conditions. More on these results here.

Those mice didn’t do too well by they way, and ended up with all kinds of heart issues. More on that study can be found here.

Then we have documents that Pfizer submitted to the Japanese health authorities, which contain biodistribution data from rat studies. Those showed that the LNPs7 could end up in all sorts of unwanted places:

Lipid distribution in rats. Graph taken from here.

One thing to note is that even with intramuscular injection, some of the lipids get into the blood, where they can quickly end up all over the place.

And here’s what was said in an assessment report for the Moderna vaccine:

Low levels of mRNA could be detected in all examined tissues except the kidney. This included heart, lung, testis and also brain tissues, indicating that the mRNA/LNP platform crossed the blood/brain barrier, although to very low levels (2-4% of the plasma level).

Maybe the “low levels” getting to the brain or heart isn’t a big deal, right? But what if we’re giving people yearly boosters?

By the way, guess how the particular ionizable lipids used in both the Moderna and Pfizer mRNA vaccines were selected (from here):

Those ionizable lipids, which are remarkably similar in structure, were discovered while the firms were optimizing LNPs for systemic administration and delivery to the liver—not the intramuscular injection of a vaccine.

I’m sorry, but when I read this, it’s just clear that this is yet another aspect of the mRNA vaccines that was rushed.

Much more can be said about the biodistribution and pharmacokinetics of the LNPs, but for now I direct you to here and here.

How about their effects on the immune system?

We already know that the LNPs can induce the release of pro-inflammatory cytokines, but they do a lot more than just that.

This paper from Sep 2022 was fascinating: Pre-exposure to mRNA-LNP inhibits adaptive immune responses and alters innate immune fitness in an inheritable fashion

They first exposed some mice to just empty LNPs. Then several weeks later they injected the mice with an mRNA vaccine encased in that same LNP. The mice that had been pre-exposed to the LNPs exhibited lower response to the vaccine, as in, lower levels of antibodies that were supposed to be elicited by the vaccine, compared to mice that hadn’t been pre-exposed to the LNPs.

This kind of thing has been known to happen sometimes; under chronic stimulation the immune system often responds with exhaustion and non-responsiveness (more here).

Since chronic inflammation can lead to immune exhaustion and non-responsiveness, we sought to determine the effects of pre-exposure to the mRNA-LNP on adaptive immune responses and innate immune fitness. We found that pre-exposure to mRNA-LNPs or LNP alone led to long-term inhibition of the adaptive immune response, which could be overcome using standard adjuvants.

Sure, the “long-term inhibition of the adaptive immune response” could be “overcome” by using adjuvants, which are substances that are sometimes included in vaccines to stimulate immune responses. But it’s alarming that pre-exposure to the LNPs could have such profound effects on the immune system. Adjuvants are just a bandaid for a problem that should be avoided in the first place. Plus, adjuvants can have their own negative side effects.

Here’s another fascinating thing:

On the other hand, we report that after pre-exposure to mRNA-LNPs, the resistance of mice to heterologous infections with influenza virus increased while resistance to Candida albicans decreased.

Candida albicans is a fungus (yeast, specifically). The mice that were pre-exposed to LNPs8 showed better resistance to influenza, but showed “significantly diminished resistance towards Candida albicans infection.”

This obviously could have implications for humans that get these types of vaccines:

If our data can be translated to humans, it is anticipated that people might present with an altered incidence of certain infections.9

Finally, from this 2021 paper:

It has been shown that mRNA-LNP vaccines have an altered tissue distribution, dynamics, and uptake in animals that have been pre-exposed to inflammatory agents. These findings suggest that people with pre-existing inflammatory conditions might show altered immune responses to these vaccines and might present with more severe side-effects.

So people with pre-existing or chronic inflammatory conditions might show altered immune responses to these vaccines. By the way, those are the very people who have been most encouraged to get vaccinated against COVID-19.

What do we know about their stability or storage requirements?

What about the stability of the LNPs or how they should be stored?

From a 2021 paper, mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability:

Currently, hardly any data is available in the public domain on what happens when mRNA-LNP formulations are stored for long periods of time. Moreover, it is unclear to what extent entrapping mRNA within LNPs influences the storage stability of the mRNA vaccine. Additionally, very little is known about the structure and morphology of LNPs formulated with mRNA, the chemical stability of the LNP components and the colloidal stability of the mRNA-LNP system.

Also, you may have heard that the Moderna vaccine needs to be kept between −15 and −25 °C whereas the BioNTech/Pfizer needs to be kept between −60 and −90 °C. Why the difference?

To date, the degradation processes and the reasons why storage temperature requirements differ, are not fully understood.

How mature is LNP technology?

To sum up:

In 2017, just a few years before the COVID-19 vaccine rollout, there were LNP researchers who thought that LNPs would go “out of fashion” because they were so inflammatory.
The LNPs are still highly inflammatory.
We don’t have a good understanding for how they unload their content into cells, including why they sometimes get stuck in endosomes inside cells.
The LNPs seem to suppress acidification of endosomes.
We think LNPs getting stuck in endosomes contributes to cytotoxicity.
We don’t have good control over where LNPs end up in the body; they’ve been shown to get into the heart, brain, ovaries, etc.
The particular ionizable lipids used in the Moderna and Pfizer vaccines were discovered while researchers were optimizing LNPs for systemic administration and delivery to the liver—not intramuscular injection of a vaccine.
Exposure to LNPs can induce responses that look like immune exhaustion.
Exposure to LNPs can alter susceptibility to different types of infections.
We don’t have much (public) data on LNP stability or storage requirements.

This only scratches the surface of the potential issues with LNPs.

There are many other aspects of LNPs that this article does not touch on. For example, we didn’t discuss the other ingredients, like polyethylene glycol (PEG), or what happens if someone is injected with LNPs that have degraded or aggregated.

When the COVID-19 mRNA vaccines were rolled out, the public was assured that mRNA technology had a “long history.” This may have led the public to think that it was a mature technology.

But a piece of technology is only as mature as its most immature component.

LNPs still have a long way to go.

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