Want to Know More About mRNA?

Ronavax Roulette: Lipid Nanoparticles (Part Two)

By Julie Beal

The ronascam is enabling a new era of synthetic biology and DNA/RNA treatments. Now the factories have been built to make the vaccines, Big BioPharma can reap vast rewards by using these factories to make an infinite number of genetic ‘therapeutics’. Vectors made in other vaccine factories are also useful to them (i.e. viruses and plasmids for DNA treatments), but mRNA is the clear favourite, because apparently it’s possible to make treatments for ANYTHING, including things that are currently ‘undruggable’.

Article Overview

This is the second part of an article about lipid nanoparticles used in the mRNA coronavirus vaccines, and it’ll take a closer look at how they can get into the brain, their potential for toxicity, and the impurities they contain. First, though, picking up from Part 1, this article will explain more about how LNPs are made, and why it matters, with a focus on the mRNA coronavirus vaccines made by Moderna and Pfizer/BionNTech, and the details contained in two reports released by the European Medicines Agency (January, 2021). Most of the details in this article will be relevant to Part 3, which is about how nanoparticles develop a protein-corona, and how this may play a part in autoimmune and/or hypersensitive reactions. Part 3 will also take a closer look at the PEG component of LNPs and how it affects the formation of a protein-corona, as well as the issue of anti-PEG antibodies. But first, let’s get the really sciencey stuff out of the way….

The MRNA LNP Formula

A four-lipid formula for mRNA-containing LNPs was worked out several years ago, and includes:

• An ionizable cationic aminolipid, such as DLin-MC3-DMA
• A phospholipid, such as DSPC
• A PEGylated lipid, such as PEG2000-DMG
• Cholesterol (it can be a proprietary blend)

The mRNA is hidden in the heart of the LNP, where it’s been complexed together with the aminolipid using an electrostatic reaction, and are formulated using rapid microfluidic mixing techniques. Cholesterol is added for structural reasons; it can be linked to the aminolipid and act as a “backbone”. Addition of cholesterol reduces the phase transition temperature of DSPC. Cholesterol can also help induce a phase transition in which the LNPs go into a hexagonal phase. The LNPs need to be hexagon-shaped to get out of the endosome of the cell, and into the cytoplasm, where these shapes also help to release the mRNA from the LNP.

The DSPC and the PEG-lipid are on the surface of the LNPs, together with a bit of the cholesterol, and a bit of the ionizable lipid.

The coronavirus vaccine made by Pfizer/BioNTech (brand name ‘Comirnaty’), and Moderna’s mRNA-1273 vaccine both have much the same mRNA code inside (for a pre-fusion version of the spike protein), and they both use much the same four-lipid formula. BioNTech have been trialling mRNA cancer vaccines since around 2012. Moderna’s first clinical trial was around 2016; they began developing their own LNPs around this time, too.

Potential Toxicity of Lipid Nanoparticles

Nanoparticles can trigger cytotoxic, genotoxic, inflammatory and oxidative stress responses in mammalian cells … Factors triggering these toxicities are not fully understood….

Potential toxicities are not fully understood because they have not been fully explored. Researchers have had ample opportunity to investigate the effects of mRNA LNPs on human health, since they have already been trialled on humans, as well as animals, but the research seems to end when they observe proteins being produced, and ‘only small changes’ in liver, spleen and blood. The thing is, if the immune system has an immediate reaction, the LNPs get destroyed and the mRNA won’t work, so the manufacturers have to try to avoid triggering certain immune responses in the initial stages. This is one of the reasons they’ve had to assess the chances of anaphylactic reactions and anti-PEG antibodies, so they do tests on blood samples in the lab. Most of the time, however, the focus is on how to get the LNPs into cells, and how to get the genetic code translated once inside. As a result, they’ve realised that various characteristics of LNPs affect the way the mRNA works, and many of these characteristics are also linked to the potential for toxicity, mainly because of the way they affect the immune system. So, when developing LNPs, they have to think about:-

• how big they are
• what they’re made of
• their net charge (e.g. + or -)
• their total surface area
• getting stuck to other particles or proteins

Way Too Fragile

It is important to recognize that the complete, intact mRNA molecule is essential to its potency as a vaccine. Even a minor degradation reaction, anywhere along a mRNA strand, can severely slow or stop proper translation performance of that strand and thus result in the incomplete expression of the target antigen.

The synthetic mRNA contained in the coronavirus vaccines is so fragile, and unstable, that it has to be kept frozen. However, mRNA can be damaged by crystallization, so some sort of antifreeze, or cryoprotectant, has to be used. It is possible to make an antifreeze using sugar (sucrose) mixed with PEG, but Moderna and Pfizer only refer to the use of sucrose. Both of the vaccines are ‘at their best’ when kept at -70°C because they begin to degrade when stored at lower temperatures. Any degradation can give rise to “lipid-related impurities” (discussed below). Small changes in temperature can also have a big effect on lipids, but little is known about the consequences of this because there is a lack of research on the freezing and storage of LNPs.

LNPs exist on a bit of a knife-edge because they can only be formed in precise conditions, and have to be maintained within tight parameters. For instance, “The method via which LNPs are synthesized is critical, because it directly affects both the LNP size and encapsulation efficiency.” LNPs are formed by condensing lipids from an ethanol solution in water, and the mixing rate has to be very carefully controlled, to try and create LNPs that are evenly dispersed, and that are of a particular size, or state.

The journey that LNPs are required to take after being injected involves a series of stages, each of which has exacting requirements. For example, they need to be the right charge at the right time. Nanoparticles are more likely to be taken up into the cell if they’re positively charged, due to the fact that the cell surface is negatively charged.  Cationic lipids have a positive charge, but are considered toxic, e.g. they can cause liver damage. They can also damage cell membranes, because “they can act as surfactants and cause membrane solubilization, poration and lysis.”

It’s as if a series of carefully choreographed moves have been designed for the mRNA LNPs, but it’s the human body that’s supposed to perform them all, by undergoing the required molecular reactions.

Spreading Round the Body – LNP Biodistribution

Whatever nanoparticles are made of, being nano-sized means they can end up in various parts of the body, including the “blood, kidney, heart, colon, bone, etc.”. This can have negative effects on cells, such as “deformation and inhibition of cell growth”. In addition to size, the way nanoparticles spread around the body depends on their surface charge (+ or – or neutral), and whether or not they’re hydrophobic (i.e. repelled by water).

The way LNPs (and the mRNA they contain) spread around the body is called biodistribution. The EMA report on the Pfizer vaccine references previous studies on similar LNPs which found they were able to get into the brain: “Several literature reports indicate that LNP-formulated RNAs can distribute rather nonspecifically to several organs such as spleen, heart, kidney, lung and brain”.

The EMA report on the Moderna vaccine also mentions the fact that LNPs can get into the brain, and describes tests conducted on animals to evaluate their biodistribution; they used a different vaccine (mRNA-1647), made with similar LNPs, to figure out how far the vaccine contents had spread around the bodies of the animals. Using evidence from a different vaccine is considered acceptable by the EMA; they’re saying that, although the mRNA genetic code was different, the LNPs were much the same, and “the distribution of the mRNA vaccine is determined by the lipid nanoparticle content”. For the tests, animals were vaccinated then ‘sacrificed’ so that various tissues could be cut up and analysed. 

According to the EMA report, 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). Liver distribution of mRNA-1647 is also evident in this study, consistent with the literature reports that liver is a common target organ of LNPs.

Hanging Around in the Body – LNP Biopersistence

Researchers who develop LNPs have identified time limits for how long they remain in the body, often referred to as the ‘half-life’. There may be a different half-life for each lipid, as well as PEG and the mRNA. In the EMA report, Pfizer say they “expect a half-life approximating 20-30 days in human for ALC-0315 and 4-5 months for 95% elimination of the lipid”.  ALC-0315 is the cationic lipid that’s been complexed together with the mRNA. Moderna’s version of this type of lipid is SM-102, which they claim is very similar to another formulation they’d already tested, called SM-86. These tests had indicated the lipid component was gone after one week.

The Blood-Brain-Barrier (BBB)

Normally, things that are injected into the bloodstream don’t reach the brain, because they can’t get through the blood-brain barrier (BBB). It’s a physical and metabolic barrier that stops most molecules getting through, but if they’re less than 200 nanometers (nm) in size, it might be possible to cross the BBB then diffuse through the extracellular space in the brain. Scientists have been trying for years to get NPs into the brain, e.g. to treat Alzheimer’s, but haven’t had much luck, partly because they can’t control where they go. However, they’re discovered that certain things help, such as controlling the size, charge, and molecular mass of the particles. For example, particles that are negatively charged will be electro-statically repelled from entering the BBB, but most of the bio-molecular drugs they want to use are high in molecular weight and polar in nature, which makes it hard to get them into the central nervous system. On the other hand, positively charged (cationic) nanoparticles get eliminated from the body more quickly than nanoparticles that are negatively charged, which has to be avoided at first so that the NPs can get into cells to translate the genetic code.

Size Matters

The size of a nanoparticle affects the way it’s processed by the body. NPs that are less than 200 nm are taken up into a cell by a process called endocytosis, but “smaller nanoparticles under 100 nm can be endocytosed more easily”. If nanoparticles are bigger than about 150 nm, they can’t get into lymph nodes (which are important for antibody production). Also, larger NPs might activate the complement system (an immune reaction that’s associated with hypersensitivity and allergy). Overall, the optimum size for a nanoparticle seems to be 100 nm or less, but NPs of this size are more of a threat to health:  “the smaller size can cause undesirable effects such as passing through the blood-brain barrier and triggering immune reactions as well as damaging cell membranes.” It would seem that as long as a nanoparticle is within a certain size range, it will be able to get through the BBB. This was shown in tests done on nanoparticles with sizes ranging from 70 nm to 345 nm; the researchers found they could all cross the BBB equally well. In other words, by the time they were that small, the particular size of the particle didn’t make much of a difference – the only difference they found was with the very smallest particles they tested, which measured 70 nm, because they got through a bit more often than the rest. Scientists have also discovered that nanoparticles less than 100 nm are able to enter BBB irrespective of their surface charge.

Moderna is not clear about the size of their LNPs, but the EMA report indicated they’re less than 161 nm (the report suggests that if they were bigger than this, they wouldn’t have been able to get into the liver of the rats they were tested on). However, a previously reported formulation created LNPs between 80 and 100 nm in size. The EMA/Pfizer report doesn’t give any details of particle size.

The Impurities in the mRNA Vaccines

This section will deal first of all with the potential impurities in the mRNA vaccines, and then with the confirmed impurities. There are a number of ways the vaccines can become contaminated, and the EMA includes details of these in its reports. For instance, the end product could contain bacterial endotoxins, picked up during production. Both companies say they’re monitoring this risk.

The EMA also raised concerns about possible nitrosamines in the vaccines (nitrosamines are classified as probable human carcinogens, and can be formed during production of a range of pharmaceuticals). Both companies have done ‘risk evaluations’, and Pfizer concluded that no risk was found.

Another issue worth looking at is the fact that neither of the vaccines contain a preservative (it stops the mRNA working as intended). Tests were done to see how long it took for microbes to grow in the vaccine vials, once they’d been opened, and apparently it took about 12 hours for them to reach significant levels. On the basis of these results, it’s been deemed acceptable to omit the preservative, although administrators are being advised to use the product within a specified time period, in order to minimize the amount of microbes getting into the vial.

In the meantime, a type of genetic material that’s confirmed as being present in both of the mRNA ronavax is dsRNA. “Double stranded RNA, also known as dsRNA, usually shows up in viruses and is somewhat unusual. In viruses, it is a unique characteristic, and only a small number of viral families exhibit this trait.” There are special immune cells which are activated by dsRNA as a defence mechanism against infection, which is why the EMA reports refer to these fragments as “potentially stimulatory”. The body may respond to it as if it’s a dangerous virus, which could set off an immune reaction that causes adverse side-effects.

There have also been some genetic mishaps whereby some of the mRNA has gone wrong during production. This means the vaccines have got random bits of RNA floating round in them as well as the mRNA that’s added deliberately. It seems to have happened in the early stages of making the mRNA, because it’s to do with how the mRNA got copied. Basically, to make synthetic mRNA, the first thing you do is make synthetic DNA! So, for example, you ‘write out’ the sequence for the rona spike, then put the DNA in some bacteria (Pfizer is using E. coli) and keep it alive in cells of some sort. The next stage is to make a copy of the DNA sequence by writing it in ‘mRNA language’ (it uses a slightly different ‘alphabet’), but it seems this process didn’t go too well, because some of the mRNA sequences were cut short. (If, say, they don’t get finished off properly with a cap and a tail, it can make the RNA unstable). The EMA/Moderna report refers to these fragments as, “short mRNA species that can occur because of abortive transcription or premature termination of transcription”. The EMA/Pfizer report uses the terms “truncated RNA” and “fragmented species”. But whatever they’re called, these novel random fragments are a worry.

But it may be more complicated than just short sequences, because Moderna said that “variants and degradants” from the mRNA sequences had become covalently linked to “impurities and degradants” in the lipids. The resulting genetic constructs are referred to as “lipid-RNA species”, and it’s said that they’re “analytically indistinguishable from unmodified mRNA and are not RNA aggregates”. It’s possible for proteins or peptides to be translated from random genetic sequences floating around, and this can be even more dodgy, but Moderna claim they can modify the lipids to stop this happening. They also say they didn’t see signs of immune cells reacting when they tested the sequences in the lab. However, the EMA said there was still “some uncertainty regarding the possible translation of additional proteins/peptides” which needed to be clarified.

Pfizer has also been asked to provide more information on the nature of the “truncated and modified mRNA species” found in their product; the EMA have recognized they might cause a cross-reaction, and have stipulated:

Relevant protein/peptide characterization data for predominant species should be provided. Any homology between translated proteins (other than the intended spike protein) and human proteins that may, due to molecular mimicry, potentially cause an autoimmune process should be evaluated.

Essentially, Pfizer is being asked to work out the molecular sequence of any rogue proteins in the vaccine, then run checks on databases to look for similar sequences in proteins that humans normally produce. If there are similarities, someone could make antibodies against the rogue protein which then attack their own body tissue as well. These are called auto-antibodies, and they’re responsible for causing auto-immune disorders. Despite their professed reservations, however, the EMA say that bits of RNA floating around are considered a necessary part of the process, i.e. ‘it’s to be expected’, so now they’re just “product-related impurities”.

All in all, lipid nanoparticles appear to be a threat to health. Using them to treat sick people with cancer and genetic diseases is a different matter entirely to using them for the entire planet. It becomes even more of an issue when the impurities are understood in relation to PEG and the protein-corona that forms around nanoparticles, as Part 3 will explore.

Read Part 1

Image Wikipedia: Lipid Nanoparticles in C Research

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For an animated description artfully explained: https://youtu.be/8iaKAWWGyWI