Design of Lipid-based Nanocarriers for mRNA Delivery

The success of COVID-19 mRNA vaccine is due to decades of research into lipid carrier delivery systems. The technology has been used to deliver a variety of bioactive molecules, such as small molecule inhibitors and vaccine components, to target cells and tissues. Lipid carrier technology has many advantages over traditional drug delivery methods, including increased drug stability, bioavailability, and distribution. Lipid nanoparticles (LNPs) are an important technology in the lipid carrier drug delivery system and have become an important development in the field of oligonucleotide-based therapies. The oligonucleotides encapsulated in lipid nanoparticles are protected from enzymatic degradation during transport and are efficiently delivered into the cell, where the contents of the carrier particles are released and translated into therapeutic proteins. Given the huge revolutionary potential of LNPs for oligonucleotide-based therapies, a new wave of researchers is pursuing more targeted applications based on LNPs.

Design of lipid nanoparticle drug carriers

Several factors should be considered when selecting lipids and how they are formulated into LNPs.

• The lipid molar ratio determines the lipid composition of particles and affects their size, polydispersity and efficacy. It is recommended to refer to similar applications that have been developed before and start from the relevant literature to determine the lipid molar ratio. The following table shows the lipid molar ratio of FDA-approved LNPs drugs:

*Ionizable cationic lipid : neutral phospholipid : cholesterol : PEGylated lipid

• The weight ratio of lipids to oligonucleotides affects the encapsulation efficiency. Most LNPs are formulated as lipids: the oligonucleotide weight ratio is 10:1.
• Ionizable lipid nitrogen: The molar ratio of oligonucleotide phosphate (N:P) indicates the charge balance between the ionizable cationic lipid cationic tertiary amine and the main anionic phosphate group of the oligonucleotide chain. This property is the basis for the complexation of ionized cationic lipids with oligonucleotides. The N:P ratio of LNP is usually around 6.
• The lipic acid dissociation constant (lipid pKa) is the pH value of the ionized and non-ionized forms of a lipid at the same concentration. Lipid pKa affects the encapsulation efficiency, efficacy, delivery and toxicity of LNP. For RNA delivery, lipid pKa is generally between 6 and 7. Specific ranges of different routes of administration have been identified. The optimal lipid pKa ranges for intravenous and muscular administration are 6.2-6.6 and 6.6-6.9, respectively.
• The three important parameters of water buffer are its composition, ionic strength and pH value. Buffer stabilized oligonucleotides in solution, ionizable cationic lipids become protonated and positively charged after mixing in acidic water buffers. The buffers commonly used in LNP preparations are 25-50 mM of sodium acetate or sodium citrate at a pH of 4-5. LNPs are dialyzed into neutral buffers such as PBS with pH 7.4 for storage and use.
• Particle size changes the pharmacokinetics of administered particles. Smaller particles typically have longer circulating half-lives because they evade clearance by mononuclear phagocytic mechanisms. Particles smaller than 100 nm can easily penetrate target tissue through porous endothelial cells. The particle size depends on the preparation method. Depending on the LNP preparation method, extrusion can be used to achieve a smaller, more uniform particle size.
• The two most common routes of administration are intravenous and intramuscular. Intravenously administered LNPS are mainly found in the liver and spleen, but also in the lungs. LNPs with net positive, neutral and negative charges can target the lung, liver and spleen, respectively. Adding cholesterol or pegylated lipids to the formula, as well as increasing the size of the LNP, increased the distribution of the spleen. Intramuscular injection is often used in vaccines because it helps lymph nodes target and activate an immune response. When a vaccine is administered, antigen presenting cells (APCs), such as macrophages and dendritic cells, are recruited to the delivery point where they can encounter the vaccine antigen. They then travel to the lymph nodes and stimulate a T cell response. It is important to note that preparations optimized for one particular route of administration are generally not suitable for other routes of administration.
• The preparation method determines the properties of LNPs, including size, uniformity and encapsulation efficiency. Cost, scalability, reproducibility and time commitment should also be considered when selecting a preparation method.

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Preparation steps of LNPs

LNP raw materials

Lipids commonly used include ionizable lipids, DSPC, cholesterol, and PEGylated lipids. Ionizable lipids are positively charged at low pH and can bind to negatively charged mRNA. Before you begin, make sure all supplies, reagents, and work environments are RNase-free. siRNA and mRNA are chemically unstable to RNase, an enzyme that degrades RNA oligonucleotides.

Generation of LNPs

LNPs are prepared by mixing a mixture of ethanol grease with an acidic water buffer containing oligonucleotides. A 1:3 ratio of the ethanol mixture to the water buffer is usually used. There are several methods suitable for small volume LNP production at the laboratory scale.

Microfluidic mixing equipment: Automated microfluidic mixing equipment or microfluidic chips are fast and efficient methods for preparing LNPs. These devices can be quickly blended in a highly controlled, repeatable manner, resulting in uniform LNPs and high package efficiency. In these devices, separate streams of ethanol lipid mixtures and oligonucleotide aqueous solutions are rapidly combined. When the lipid nanoparticles are formed, the two strands of solution are mixed and collected into a separate tube. LNPs can be fine-tuned by changing parameters such as flow ratio and total flow rate.

T-shaped or Y-shaped mixers: These mixers can be assembled from common and affordable laboratory materials. The T or y joint can be fitted with two inlets connected to a separate syringe containing a lipid mixture or oligonucleotide solution, one outlet guides the LNPs into the collection tube, and the inlet flow can control the injection pump.

Ethanol injection: This method is suitable for all laboratories. The mixing of the ethanol mixture and the oligonucleotide aqueous solution is carried out with the help of a magnetic stirring plate. The ethanol mixture was injected into the acidic oligonucleotide solution and stirred continuously for 30 minutes. However, this method may produce more non-uniform LNPs, with lower encapsulation efficiency and easy to change.

Hand mixing: This is a simpler alternative to ethanol injections. The ethanolipid mixture was transferred to an acidic oligonucleotide solution and mixed by rapid pipetting for 15 seconds. Let the mixture sit for 10 minutes. As with the ethanol injection method, LNPs mixed by hand obtained heterogeneous LNPs with low encapsulation efficiency, and the results were variable.

Final preparation steps of LNPs

The final preparation of LNPs is carried out after formation in the mixing step. The following steps ensure that these wastes are homogeneous, stable and free of any residual chemical or biological contaminants during storage and use.

Extrusion: Extrusion reduces particle size and produces a uniform particle size distribution. This step is usually performed with a large number of mixing methods, such as ethanol injection and hand mixing methods. Dialysis: LNPs are dialyzed in storage buffers using appropriate molecular weight truncation (MWCO) tubes. This step removes unencapsulated cargo, excess lipid components and ethanol from the final preparation. Dialysis can also regulate the pH of LNPs from acidic preparation buffer to neutral storage solution.

Filtration disinfection: Filtration is the recommended method for LNPs sterilization. Sterilize LNPs with a 0.22 μm filter before storage to remove bacteria or other contaminants. For larger particles or highly viscous solutions, other sterilization methods such as autoclaving or irradiation may be employed, although these methods may affect the structural integrity of the Lingtu nuclear power plant.

Optimization of mRNA-LNP physicochemical properties

As an advanced gene delivery platform, mRNA-LNPs show great potential in the field of vaccines and therapeutics. Optimizing its physicochemical properties is critical to improving delivery efficiency and in vivo performance, with key parameters including particle size, surface charge, encapsulation efficiency, serum stability, and half-life.

Particle size and surface charge

Particle size and surface charge significantly affect the delivery efficiency of mRNA-LNPs. LNPs with particle size in the range of 50-150 nm can effectively avoid rapid clearance by the liver and kidney and enhance tissue penetration. Surface charge affects cell uptake and biological distribution. Moderate positive charge can promote the interaction with membrane anionic phospholipids and improve the efficiency of transduction, but too strong positive charge may cause toxicity, so it is necessary to balance efficiency and safety.

Package efficiency

Efficient mRNA encapsulation is essential for the stability and functional release of LNPs. Fine-tuning the proportions of LNPs components, such as ionized lipids, structural lipids, cholesterol, and PEG lipids, enables optimal mRNA protection and targeted intracellular release, thereby preventing mRNA degradation and ensuring its efficient expression.

Serum stability and half-life

Serum stability and half-life determine whether mRNA-LNPs can successfully reach targeted tissues. Optimizing the composition and surface modifications of LNPs, such as PEG treatment, can enhance their ability to fight nucleases and other degrading enzymes in serum, extend cycle times, and improve delivery efficiency.

LNPs stability and storage

The composition of LNPs has a decisive influence on its stability. LNPs are usually composed of ionized lipids, structural lipids, cholesterol and pegylated lipids. Ionized lipids can better maintain the stability of LNPs in acidic environment, while cholesterol can improve its stability and performance in physiological environment. In addition, pegylated lipids can not only increase the cycle time of LNPs, but also reduce their accumulation and precipitation during storage.

LNPs storage conditions directly affect its stability. Typically, LNPs must be stored at low temperatures to slow chemical degradation and physical instability. Freeze-drying technology is often used to convert LNPs from liquid to solid to extend their shelf life and stability. At the same time, preventing long-term light and oxidation is also a key measure to ensure the stability of LNPs.

In addition, pH value and ionic strength also affect the stability of LNPs. Deviations from neutral pH or high ionic strength environments may cause lipid degradation or rearrangement in LNPs. Therefore, the storage solution of LNPs is usually adjusted to the appropriate pH value, and deionized water or weak buffers are used as much as possible.