An Introduction to mRNA-LNP Formulation Technology

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What is mRNA-LNP?

mRNA is a single-stranded ribonucleic acid molecule that carries genetic information, with its primary function inside cells being to transmit genetic information from DNA to ribosomes, thereby directing protein synthesis. This characteristic of mRNA makes it a key molecule in gene therapy and vaccine development. Lipid Nanoparticles (LNPs) are nanoscale particles composed of a lipid bilayer, exhibiting good biocompatibility and biodegradability. Their main components include phospholipids, cholesterol, among others, which can naturally degrade in the body, thereby reducing potential toxicity. By encapsulating mRNA within LNPs, scientists can effectively protect mRNA from degradation by intracellular enzymes, ensuring its stable delivery to target cells. This process involves electrostatic interactions between mRNA and LNPs, as well as the encapsulation of mRNA by the lipid bilayer of LNPs.

The schematic diagram of COVID-19 mRNA-LNP vaccines.The schematic diagram of COVID-19 mRNA-LNP vaccines.

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Types of mRNA Vaccines

The core principle of mRNA vaccines is to encode antigenic genetic information within mRNA, deliver it to the host cell cytoplasm, express it in vivo, and induce antigen-specific immune responses. mRNA vaccines can produce pathogens targeting any known protein target, and mRNA is produced using cell-free enzymatic transcription reactions, ensuring rapid amplification for vaccine production. Currently, there are three main types of RNA vaccines: mRNA, bmRNA, and saRNA.

Compared to non-amplified RNA, the main advantage of self-amplifying RNA is self-replication, requiring only low doses of RNA for rapid amplification without cellular involvement. This significantly reduces costs for large-scale production and allows for clinical application in a short period.

Key Excipients of Lipid Nanoparticles (LNP)

Cationic Lipids

Cationic lipids are key components of LNP delivery systems, capable of efficiently binding with negatively charged mRNA and effectively encapsulating nucleic acid drugs. Simultaneously, they provide a positive charge which, upon complexing with negatively charged mRNA, aids in endosomal escape and intracellular transfection. Cationic lipids also exhibit pH sensitivity; upon cellular uptake, LNPs fuse with the low pH environment of the endosome, releasing mRNA into the cytoplasm. Developing cationic lipids requires balancing delivery efficiency with cytotoxicity. The cytotoxicity of cationic lipids depends on the structure of their hydrophilic headgroups; for instance, amphiphilic molecules containing quaternary ammonium headgroups are more toxic than those containing tertiary amines.


Cholesterol primarily facilitates LNP endocytosis and helps maintain the bilayer structure and fluidity of lipids within LNPs. Auxiliary neutral lipids (such as various phospholipids) are also used to construct the LNP bilayer structure, as the bilayer structure of cationic lipids is unstable.

Helper Lipids

Representative auxiliary lipids include DOPE, DSPC, and DOPC, among other phospholipids. In the process of preparing cationic liposomes, helper lipids play a strong synergistic role, mainly stabilizing the bilayer membrane and reducing the toxicity of positive components. They promote mRNA release upon LNP endocytosis, assist in the cellular penetration of cationic liposomes, determine the morphology of mRNA-LNP complexes, ensuring good fusogenicity, and enhancing transmembrane efficiency.

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PEGylated Phospholipids

Liposomes modified with specific structured PEG can control the particle size during nanoparticle synthesis. Due to the strong hydration of the PEG ethylene oxide chain segment, the PEG structure can form a hydrophilic protective layer in the aqueous phase, effectively preventing nanoparticle aggregation during storage and maintaining the spatial stability of LNPs. Additionally, PEG on the surface of LNPs protects the particles from detection by immune proteins in the body, shields against the binding of plasma proteins, and prevents LNP clearance in the body.

Stabilizers (Such as Sucrose or Trehalose)

The addition of stabilizers enhances the stability of LNPs and mRNA vaccines, prevents excessive lipid viscosity, and provides delivery efficiency.

Principle of mRNA-LNP Delivery

(1) Before cellular uptake, cationic lipids can electrostatically complex with negatively charged mRNA molecules, forming complexes that enhance the stability of mRNA molecules.

(2) Upon reaching the cell membrane, cationic phospholipids trigger membrane fusion with negatively charged cell membranes, destabilizing the cell membrane and facilitating mRNA delivery.

(3) Upon internalization into the cell, lysosomes containing various hydrolytic enzymes degrade exogenous and endogenous macromolecules, lowering the pH and creating an acidic environment. This protonates the ionizable lipids, disrupting the bilayer structure of LNPs and releasing mRNA. Following the central dogma, mRNA binds to ribosomes responsible for protein synthesis, translating into viral proteins, like antibodies, which neutralize the virus.

Preparation Methods of mRNA-LNP

Common methods for preparing lipid nanoparticles include thin-film hydration, extrusion, and homogenization. Currently, advanced microfluidic mixing techniques are used to prepare nucleic acid lipid nanoparticles, which are relatively simple, fast, and mild, while being easily scalable for production. Through microfluidic machines of different production scales, the preparation of mRNA-LNP can be achieved consistently, with appropriate particle sizes and extremely low PDI values, confirming good dispersibility and encapsulation efficiency of over 90%. With efficient and scalable preparation of nucleic acid lipid nanoparticles using microfluidic technology, production rates can reach up to about 20 liters per hour.

Basic Principles of Microfluidic Technology

After dissolving lipids and nucleic acids separately in aqueous and organic phases, the two-phase solutions are injected into the system through two inlet channels. One end contains the RNA aqueous solution, while the other contains the lipid ethanol solution. Through rapid mixing of the two phases, the synthesis of nucleic acid lipid nanoparticles is completed. By altering the fluid injection rate and ratio, the particle size of lipid nanoparticles can be controlled.

Impact Jet Mixing Method

This method enables large-scale production by using a high-pressure pump to create two jets of vaccine and lipid solutions, which collide in the chamber. Fluid dynamics are utilized to thoroughly mix the lipid components, forming nano-sized LNPs encapsulating mRNA. The collision jet mixer is installed in a cleanroom (Class C), with flow rates of all units controlled through a central control platform. All units are installed within a stainless-steel frame to accommodate CIP cleaning procedures in drug production. The system includes: a KNAUER collision jet mixer (IJM), inlet pipelines for lipid/ethanol mixtures and mRNA/buffer mixtures, an outlet pipeline for liposomes, a CIP online cleaning system, and a stainless-steel frame.

Applications of mRNA-LNP

Gene Therapy: A Novel Approach to Disease Treatment

mRNA-LNP technology offers a new avenue for gene therapy. By designing specific mRNA sequences, scientists can synthesize therapeutic proteins to repair or replace damaged genes. This approach holds promise for the treatment of various genetic and rare diseases.

Vaccine Development: Rapid, Efficient Vaccine Production Method

mRNA-LNP technology holds significant advantages in vaccine development. Compared to traditional vaccines, mRNA vaccines' production process is faster, more efficient, and easily scalable. Additionally, mRNA vaccines exhibit strong immunogenicity, inducing long-lasting immune protection. Therefore, mRNA-LNP technology has become the preferred platform for novel vaccine development.

Drug Delivery Systems: Enhancing Drug Efficacy, Reducing Side Effects

mRNA-LNP serves as a drug delivery system with broad application prospects. By encapsulating drugs within LNPs, scientists can achieve targeted drug delivery, enhancing efficacy and reducing side effects. This method holds promise in cancer treatment, anti-inflammatory drug delivery, and other fields.

Advantages of mRNA-LNP Compared to Other Technologies

Safety: Reduced Potential for Immune Reactions and Toxicity

Compared to traditional gene therapy and vaccine development technologies, mRNA-LNP technology offers higher safety. Since mRNA does not integrate into the host genome, it reduces the potential for immune reactions and toxicity risks. Additionally, the lipid bilayer of LNPs can naturally degrade in the body, further enhancing the safety of this technology.

Efficiency: Enhanced Gene Expression and Protein Production

mRNA-LNP technology demonstrates significant efficiency. By encapsulating mRNA within LNPs, scientists can protect mRNA from degradation and enhance its stability within cells. This helps improve efficiency in gene expression and protein production, thereby enhancing therapeutic effects.

Scalability: Adaptable to Various Disease Treatment and Prevention Needs

mRNA-LNP technology possesses strong scalability. By designing different mRNA sequences and optimizing LNP compositions, scientists can develop treatment and prevention strategies for various diseases. This flexibility makes mRNA-LNP technology a crucial tool for treating and preventing multiple diseases in the future.

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