# Introduction
## Overview
Update on the current state of evidence RE mRNA lipid nanoparticle vaccines
- how they distribute
- how long they persist
- how long they are expressed
- what this all leads to

## Early Reports
- January 2020 - antibodies to detect Nucleocaspid protein of SARS-CoV-2
*Usually takes a while (supposedly had come into existence or had been isolated in the beginning of 2020 (Corman Drosten PCR) to produce a nucleocaspid antigen in some form (PCR and express it in some cells), immunize animals, extract antibodies from those animals to use this antibody in immunohistochemistry*
- 2006 (also RT-PCT assay of SARS-CoV-2)

# Distribution
*A study which Pfizer submitted to regulatory authorities which came out into the open through Japanese regulators.*
Pfizer used a model protein (Luciferase) in place of SARS-CoV-2 S protein. Lipids made radioactive. 
- Injected rats
- Follow marked lipids
## Results
Rapidly, the vaccine shows up in the blood plasma (15 minutes onward). After injection, there is a measurable level in the blood plasma which peaks for 2 hours and concomitantly the labelled vaccine accumulates in various organs:
  - high levels in the liver and spleen
  - Also in the ovaries and adrenal glands 
  - Testes
  - Levels were still increasing in some organs at extent of this (no idea what peak would be)

## Questions
How can we understand this particular pattern of distribution between different organs. Will discuss in details how the lipid nanoparticles are supposed to work
### Details
- Mixture of 4 different lipids
- wrap modRNA (chemically modified mRNA - more stable - proline mutations)
- Ionizable lipid which at proper pH is cationic which wraps the negatively charged mRNA
- 2 naturally-occurring (cholesterol and phosphatidylcholine) stabilize structure of lipid nanoparticle
- Surface has Polyethylene glycol-linked lipid
  - Gives particle and overall hydrophilic / water-soluble characteristic
- Cholesterol leaks out from this particles, meaning they are no very stale. Cholesterol leaves and crystallizes on its own (this modifies the activity of these particles)

### Molecular Structures
*2 major synthetic lipids (ALC for Pfizer vaccines, for example)*
- In both cases we have 2 acetyl chains (aklyl chains in one case) with 14 carbons
  - This length judiciously chosen: enables lipids to loosely attach with LNPs, which affects in-vivo behaviour

#### Loose Lipids
In a nutshell, PEGylated lipids are replaced with something from the subject, such as cholesterol
- Pegylated lipids desorb from the particle, exposing less H2O-soluble lipids underneath
- Stabilize by binding with protein
  - Apolipoprotein (wraps lipid transport particles - lipoproteins)
  - Other proteins: Ab and complement proteins

### Apolipoproteins
Q. What is the function of these particles?
A. Mediate the transport of lipoproteins or, in this case, LNPs into cells and in across cell barriers

- Produced in liver and small intestine
- Overcomes cellular behaviour in blood vessels using transcytosis
  - Cells receptors recognize apolipoproteins
  - Release particles on the tissue side

## What This Means
### Biomolecular Corona
- LNPs will accumulate in organs which allow apolipoprotein
  - Liver (produces)
  - Endocrine glands (cholesterol used as precursor for hormone synthesis)
  - Placenta accumulates for progestin production and to supply lipoprotein to fetus
  - Lactating breast glands acquire fat/cholesterol from lipoproteins
  *Also expect spleen/lymphatic organs to accumulate LNP*

## Intracellular Fate of mRNA/LNP
- Endocytosis (perhaps via apolipoprotein receptor)
- Endosome (vessicle encasing the particle) is acidified