Lipid Nanoparticle

Messenger RNA (mRNA) has flourished as a strategy to generate transient gene expression in immune cells to mitigate the disadvantages associated with viral vectors, including CAR therapy. Lipid nanoparticles for mRNA delivery (LNP) have been used in the pharmaceutical industry as vectors for the delivery of a variety of therapeutics. BOC Sciences' platform offers fully validated mRNA delivery or mRNA LNP delivery systems to induce functional protein expression and to investigate the potential of mRNA and LNP-based gene therapy.

Introduction

Composition and Structure of mRNA/LNP

In addition to negatively charged mRNA, Lipid nanoparticles (LNP) loaded with mRNA have four other components: ionizable cationic phospholipids, neutral auxiliary phospholipids, cholesterol, and polyethylene glycol modification PEGylated lipid)

Components	Proportion
Cationic phospholipids (ionizable)	About 50%
Auxiliary phospholipids (neutral)	About 10%
Cholesterol	About 38.5%
PEGylated phospholipids	About 1.5%

Table. 1 Proportion of LNP components

The role of excipients in lipid nanoparticles is similar to that of such excipients in liposomes:

Neutral auxiliary phospholipids, generally saturated phospholipids, increase the phase transition temperature of cationic liposomes, support the formation of lamellar lipid bilayers and stabilize their structural arrangement.
Cholesterol has strong membrane fusion properties and promotes intracellular uptake and cytoplasmic entry of mRNA.
PEGylated phospholipids are located on the surface of the lipid nanoparticles, improving their hydrophilicity, avoiding rapid clearance by the immune system, preventing particle aggregation and increasing stability.
The most critical excipient is the ionizable cationic phospholipid, which is a decisive factor in mRNA delivery and transfection efficiency.

Delivery of mRNA/LNP

Ionizable cationic phospholipids

Ionizable cationic phospholipid is a key determinant of mRNA delivery and transfection efficiency. The cationic phospholipid needs to be non-ionized under physiological conditions (pH=7.4) and ionized under acidic conditions (≤5.0).

Principle of mRNA/LNP delivery

Before entry into the cell, cationic lipids can achieve electrostatic complexation with negatively charged mRNA molecules, forming a complex that improves the stability of the mRNA molecules. When mRNA/LNP reaches the cell membrane, the cationic phospholipid triggers membrane fusion with the negatively charged cell membrane, which destabilizes and facilitates the delivery of mRNA molecules. After internalization into the cell, as the lysosomes containing a variety of hydrolytic enzymes break down the exogenous and exogenous macromolecules, the pH drops to create an acidic environment, which protonates the ionizable lipids and disrupts the bilayer structure of the LNP, releasing the mRNA, which binds to the ribosomes responsible for protein production in accordance with the 'central law' and is translated into viral proteins, i.e. antibodies, which neutralize the virus.

Advantages of LNP for mRNA Delivery

Efficient nucleic acid encapsulation and efficient transfection
Improved tissue permeability for therapeutic delivery
Low cytotoxicity and immunogenicity

Applications of LNP-mRNA Delivery

Vaccine development
In vivo antibody delivery
Chimeric antigen receptor (CAR) T/NK generation
Protein replacement therapy
Immune cell engineering: macrophages, T cells, NK cells, B cells, dendritic cells, Gamma Delta T cells

Fig. 1 Schematic representation of the different types of lipid-base nanovectors (Guevara ML,2020)