Lipid Nanoparticles - How do they work - Structure of LNPs - LNPs in mRNA vaccine Pfizer/Moderna

Описание: In this video, Dr. Aizaz from Medicovisual describes how Lipid Nanoparticles work and what is their structure.
Previously we have already discussed how mRNA vaccines work. The idea is to transport the mRNA of the spike protein of coronavirus into the cell. The cell will then create spike proteins and will train the immune system to recognize the Sars-CoV-2 from its spike protein.
The problem here is that a naked RNA cannot enter into the cell. And of course, without entry into the cell, the mRNA vaccine will never work. Furthermore, the RNA particles present outside the cell will be quickly degraded by the RNAses present in the extracellular environment.
To solve this problem, Lipid nanoparticles were created. These are incredibly small particles, measured at a nanoscale. [From your secondary school knowledge, you must be knowing that 1 nm = 10-9m]. These tiny lipid nanoparticles are made up of fat/lipids. They can keep the RNA safe from the RNAses and can faithfully transport the RNA inside the cell, into its cytosol.
But the question is why does mRNA, which is a type of RNA by the way, cannot, by itself, cross the cell membrane and enter into the cell? Why does it need external help from either a viral vector or a lipid nanoparticle?
The reason is that RNA [and DNA too] is negatively charged, because of the presence of negatively charged Phosphate ions attached with each of its structural subunits; nucleotides. So, it is the Phosphate ions that impart a net negative charge to the RNA and DNA.
Charged or ionic substances have a hard time crossing the cell membrane made up of lipid bilayer.
When surrounded by the lipid nanoparticle, RNA can cross the lipid-bilayered cell membrane as it becomes a lipophilic substance.
You can imagine it from a simple analogy. Let us suppose you want to a restricted area where only Police is allowed to go. Of course, you will be denied entry at the gate. [Think of the gate as the cell membrane].
You still want to go to that restricted area. You call your friend who is a police officer. You sit in his police van and then easily pass through the security checkpoint.
The same is happening here. Foreign RNA is not allowed to go inside, so it enters into this police van or LNP to cross the cell membrane.
After understanding why there is a need for a lipid nanoparticle. Let us now discuss what is it made up of and then will understand how it works.
Structure and components of LNPs
As discussed earlier, RNA is negatively charged. You must be aware of the fact that opposite charges attract. So, cationic lipids [positively charged ions] will attract and thus bind with the negatively charged [Anionic] RNA.

Cationic lipids contain an amine group head that has a positive charge on it. Along with that, it has legs made up of fatty acid chains, that are non-polar or non-ionic.
When such cationic lipids come across the RNA, they will obviously orient their charged [cationic] amine head towards the RNA and will orient the legs outward.
Permanently cationic [having a permanent positive charge] lipids are toxic and can disrupt the integrity of cell membrane [1]
To mitigate this problem, ionizable cationic lipids are used. These lipids are either neutral or slightly charged [cationic] at physiological pH. But in the lab, they create the vaccine at an acidic pH.[2] On acidic pH, the Proton or H+ ion quickly attaches with the amine head of the lipid causing it to acquire a positive charge. Due to this positive charge, it embraces and encapsulates the negatively charged RNA.

At physiological pH, as these ionizeable cationic lipids will become neutral, they may cease to embrace the RNA. So this particle is further surrounded by one or more additional layers of phospholipids. Thus RNA will still remain packed and protected inside the core of lipid nanoparticle.
They are mostly non-cationic structural lipids with some cationic lipids too embedded in between them, here and there.

Then there are some lipids, conjugated with a special molecule called PEG [Polyethylene glycol], that too are embedded into this outer layer. The outer layer of PEG prevents the aggregation or fusion of LNP particles with each other due to its steric hindrance [3]
Polyethylene glycol offers a steric hindrance and prevents the immediate take-up of these lipid nanoparticles by immune cells followed by their destruction. [3] Thus, it slows down the clearance of these LNPs quickly from the body.
PEGylation is reversible in such a way that LNPs gradually undergo de-PEGylation. PEG is gradually removed and the de-PEGylated LNP is then picked up by the cells gradually.
Cholesterol is also present interspersed in the spaces between the nanoparticles. It maintains the integrity and imparts the structure stability to LNP. Cholesterol, and structural phospholipids, forming the outer layer of LNP, are also called helper lipids

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