mRNA (messenger RNA), is a type of single-stranded RNA that is transcribed from a strand of DNA as a template, which carries genetic information and can guide protein synthesis. mRNA was first proposed by Jacques Monod and François Jacob, and later discovered by Jacob, Sydney Brenner and Matthew Meselson at Caltech in 1961. Since 2015, the mRNA technology has been growing at an accelerated pace as delivery and modification technologies have matured, and biotechnology companies such as CureVac, BioNTech, and Moderna, which are focused on mRNA technology, have come to prominence. The COVID-19 pandemic has brought mRNA vaccines and drugs to widespread attention worldwide and has led to a boom in research in the mRNA field. According to Nature related statistics, as of the end of July 2021, there are 180 mRNA vaccine and drug pipelines in development worldwide, the vast majority of which are related to infectious diseases, rare diseases and oncology.
mRNA has high molecular weight and strong hydrophilicity, but its single-stranded structure makes it extremely unstable and susceptible to degradation. The limited lifetime of mRNA allows cells to rapidly change protein synthesis in response to its changing needs, but it is difficult to meet the requirements of druggability. In addition, mRNA molecules carry a negative charge and have difficulty crossing the cell membrane with the same negative charge on the surface, so special modification or wrapping delivery system is required to achieve intracellular expression of mRNA drugs, therefore, delivery technology is one of the core patented technologies of mRNA companies.
Lipid Nanoparticle (LNP) is currently the dominant delivery system. They are often used in vaccines due to their relatively easy uptake by antigen-presenting cells. The three major mRNA vaccine giants Moderna, CureVac and BioNTech are currently using LNP delivery technology for their COVID-19 vaccines.
Structure of Lipid Nanoparticles
The LNP components that are widely used at this stage include the following four major categories: cationic or ionizable lipids, cholesterol, PEGylated lipids and phospholipids.
1. Cationic Or Ionizable Lipids
Cationic lipids (CLs) and ionizable lipids (ILs) can initiate the first step of self-assembly through electrostatic interactions. Lipid complexes containing cationic lipids are still widely used for nucleic acid delivery. However, they have been largely replaced by pH-responsive ionizable lipids due to toxicity concerns and lack of in vivo potency. ionizable lipids in LNP formulations behave as neutral at physiological pH, while positively charged in the acidic environment of endosomes. The pH-dependent ionization ability makes ionizable lipids suitable materials for nucleic acid delivery due to the large improvement in potency and toxicity characteristics.
The development of cationic lipids requires a balance between delivery efficiency and cytotoxicity. The cytotoxicity of cationic lipids depends on the structure of their hydrophilic head groups, e.g., amphiphilic molecules containing quaternary ammonium head groups are more toxic than amphiphilic molecules containing tertiary amines. Therefore, addressing the cytotoxicity of ionizable cationic lipids is one of the key points of LNP technology and the focus of patent protection. The proprietary cationic lipids ALC-0315 (Acuitas/Pfizer) and SM-102 (Moderna) both have hydrophilic head groups that are tertiary amines that can be positively charged by protonation in a physiological low pH environment and are safely cleared after mRNA is delivered.
Cholesterol is often included as a helper that improves intracellular delivery as well as LNP stability in vivo. Helper neutral lipids (e.g., various phospholipids) are also used to construct LNP bilayers because the bilayer structure of cationic liposomes is not stable.
3. PEGylated Lipids
LNPs modified by PEG with specific structure can control the particle size of nanoparticles during nanoparticle synthesis. Due to the strong hydration of polyethylene glycol ethoxy links, the PEG structure can form a hydrophilic protective layer in the aqueous phase, which can effectively prevent the aggregation of nanoparticles during storage, thus maintaining the spatial stability of LNP. At the same time, PEG on the surface of LNP particles can protect the particles from being detected by immune proteins in vivo, shield the particles from being bound by plasma proteins and other components, and prevent LNP particles from being cleared in vivo. The proprietary PEG lipid structure ALC-0159 (M-DTDAM-2000) currently selected by Pfizer and M-DMG-2000 selected by Moderna are both derivatives of PEG-2000.
Helper lipids are represented by phospholipids such as DOPE, DSPC and DOPC. In the preparation of cationic liposomes, helper lipids have very strong synergistic effects, mainly including stabilizing bilayer membranes and reducing the toxicity of cationic components, promoting the release of mRNA when LNP is endocytosed and assisting the cell permeation of cationic liposomes, and determining the morphology of mRNA-LNP complexes, making the complexes well fusible and improving the transmembrane efficiency.
Huateng Pharma has the capability to produce LNPs delivery system excipients on a large scale and has been supplying a wide range of PEG lipids and helper lipids, including mPEG2000-DMG and ALC-0159, to domestic and international customers. Huateng Pharma is an industry leader in impurity content, purity, batch-to-batch stability, analytical capability, and customization.