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Lipid Nanoparticles (LNPs) Design and Formulation Production

Compared with viral vectors, non-viral delivery vectors are easier to scale up and have the ability to solve the most critical safety issues. Thanks to the LNP-mRNA vaccine that came to the fore during the COVID-19 pandemic, lipid nanoparticles (LNP) are undoubtedly the most advanced and widely studied non-viral vectors at present. Typically, LNP is composed of 4 components: ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Each component determines the stability, transfection efficiency, and safety of LNP. It plays a vital role. To design a drug carrier based on lipid nanoparticles, we first need to clarify the basic principles behind LNP design.

Physicochemical Characteristics to Consider When Designing LNP

  • Formulation: Molar Ratio, Weight Ratio and N:P

The lipid molar ratio determines the composition of the lipid particles and affects their size, polydispersity, and efficacy. However, an important feature that distinguishes LNPs from lipoplexes is that their structure cannot be predicted simply from the molar composition of the formulation. LNP needs to be characterized by cryo-electron microscopy and other characterization methods to characterize the LNP structure under different formulas.

Changes in the content of different components will not only affect the morphology of LNP, but also affect the encapsulation efficacy. Studies have found that when the ICL molar ratio increases, the cholesterol content decreases proportionally, which will lead to a decrease in the encapsulation efficiency of loaded nucleic acids. Cholesterol is important for the interaction between ICL and nucleic acids, so cholesterol deficiency results in reduced encapsulation efficiency. In addition, if the formulation contains a higher proportion of PEG lipids (>2.5 mol%), the encapsulation efficiency will be significantly reduced. At the same time, due to the commonly found human anti-PEG antibody response, lower PEG lipid ratios are often considered in formulation design, or are gradually replaced by analogues.

  • Fatty Acid Dissociation Constant (pKa)

The pKa of ionizable cationic lipids (ICL) determines the ionization behavior and surface charge of LNPs, and further affects the stability and toxicity of LNPs. Traditional permanently charged cationic lipids such as DOTAP (trimethyl-2, 3-dioleoyloxypropyl ammonium bromide), which were used in earlier studies for nucleic acid delivery, easily interact with negatively charged serum proteins in the body. The occurrence of aggregation will lead to rapid clearance of LNP by the mononuclear phagocyte system, seriously shortening the half-life of LNP in vivo. And this aggregation effect increases the risk of toxic side effects, leading to red blood cell membrane damage and hemolysis.

Ideally, at acidic pH (i.e., the endosomal environment), the head group of the ICL should become positively charged again to facilitate binding to negatively charged lipids exposed on the endosomal membrane. This interaction causes the lipid carrier to form an inverted hexagonal structure, further promoting the destruction of the proendosomal membrane and delivering therapeutic nucleic acids into the cytoplasm. For example, studies have found that LNP pKa values of 6.2-6.5 and 6.6-6.9 are beneficial for in vivo hepatic delivery of siRNA and intramuscular injection of mRNA vaccines, respectively.

  • Physicochemical Properties

The physicochemical properties of LNPs, including size, surface charge (zeta potential), and surface modifications, have a direct impact on the efficacy, pharmacokinetics, and biodistribution of LNPs. LNP size is critical to overall drug targeting and circulation longevity. Studies have shown that smaller particles are more likely to evade clearance by mononuclear phagocyte machinery and generally have a longer circulating half-life. In addition, particles smaller than 100 nm can easily penetrate target tissues through porous endothelial cells. In addition to different particle sizes caused by formula changes, different LNP preparation methods can also be used, such as extrusion to achieve smaller and more uniform particle sizes.

The Main Components and Principles of LNP

  • Ionizable Cationic Lipids(ICLs)

Ionizable cationic lipids are key components in LNP formulations and have an amphiphilic structure with a positively charged hydrophilic amine head linked to a long lipophilic tail. The structure of ICLs can be divided into amine headgroups (Headgroups), hydrophobic tail groups (Tails) and internal linking segments (Linkers).

Depending on the number of amine groups in the head group, cationic lipids can be divided into monoamine-based or polyamine-based lipids. The most famous DLin-MC3-DMA (MC3), SM-102 and ALC-0315 are all monoamine-based lipids and are the only three ionizable cationic lipids approved by the FDA for RNA delivery. However, these three ionizable cationic lipids are not biodegradable, and accumulation in the body will produce potential cytotoxicity. Researchers often focus on tuning the structure of lipid tails to enhance potency or confer specific functions by changing the number of tails, designing linear or branched structures, and introducing unsaturated or biodegradable bonds. For example, the unsaturated tail of MC3 promotes endosomal escape of siRNA, and the ester bond of L319 can accelerate the degradation of intracellular lipids.

  • Phospholipids

Phospholipids are accessory lipids that assist lipid nanoparticles in self-assembly and endosomal escape. In preclinical research and clinical applications, commonly used phospholipids are DSPC and DOPE. DOPE is a phosphoethanolamine with two unsaturated chains (C18). Due to its unsaturated nature, DOPE has clotogenic characteristics and therefore promotes the fusion of LNPs with endosomal membranes by promoting a non-bilayer inverted hexagonal structure. In contrast, DSPC is a saturated amphipathic lipid. It has a neutral overall charge and consists of a quaternary amine and a negatively charged phosphate group connecting two saturated chains (C18). Due to the saturation characteristics, DPSC has a cylindrical shape and therefore does not exhibit membrane instability properties but tends to form a stable bilayer structure. The two mRNA COVID-19 vaccines already on the market, Moderna and BioNTech/Pfizer, both use DPSC.

  • Cholesterol

Cholesterol helps increase the stability of LNPs and assists in cell membrane fusion. Optimizing the structure of cholesterol can also improve the delivery efficiency of LNPs and give LNPs specific functions. Some studies have screened β-sitosterol LNP (eLNP) among natural cholesterol analogs, which significantly improves transfection efficiency. By analyzing the SAR of this cholesterol analog, they found that eLNP has a polyhedral structure and is composed of different surface lipids. This may facilitate endosomal escape and mRNA release.

Pegylated (PEG) lipids are mainly used to reduce nanoparticle aggregation, reduce phagocytosis by mononuclear phagocytes, and prolong systemic circulation time. However, PEG lipids can also hinder target cell interaction and endosomal escape, thereby reducing transfection efficiency. By optimizing the PEGylation chemistry and the density of PEG on the LNP surface, the pharmacokinetics and pharmacodynamics of LNPs can be altered.

Research data shows that the shorter the PEG lipid carbon chain, the faster the desorption rate. The length of the dialkyl chain and PEGylated lipid concentration/molecular weight significantly affect the pharmacokinetics, pharmacodynamics, and biodistribution of LNPs in vivo. PEG that is too short (<1 kDa) cannot prevent the interaction of nanoparticles with serum proteins and therefore cannot effectively extend circulation time. However, very long PEG (>5 kDa) or high molar ratio of PEG (>15 mol%) will lead to reduced membrane permeability, thereby severely reducing cellular uptake. Therefore, PEG molecules with medium molecular weight (∼2 kDa) are usually used for LNP modification at a proportion of <5%.

Stability and Storage of LNPs

Stability studies are critical in helping to determine the shelf life and optimal storage conditions of the final product. The stability of LNP-nucleic acid formulations depends on processing parameters such as temperature, humidity, and light, as well as the properties of the nucleic acid and lipid excipients. It is known that commercially available mRNA LNP vaccines are frozen due to lack of long-term stability in solutions at 2-8°C. The limited shelf life under refrigerated conditions is mainly attributed to the chemical degradation of the mRNA and the reactive properties of nucleotide and lipid impurities in solution.

Methods to improve LNP-RNA stability include formulation strategies such as the addition of buffers, surfactants, and other excipients. In addition, the use of effective process controls and the use of appropriate cryoprotectants (sucrose, trehalose or mannitol) for freezing or lyophilization are often used. Although this process is known to affect LNP size and encapsulation, these properties are not the only determinants of LNP performance. Furthermore, the choice of excipients in solution (e.g., buffers and sugars) is crucial for this method, indicating that the solution medium has a strong influence on the formation and colloidal stability of LNPs upon reconstitution. The colloidal stability of LNPs will be affected by their lipid composition, suggesting that efficient lipid mixing and interaction with RNA cargo is necessary to obtain a more stable product.


  1. Cárdenas, al. Review of structural design guiding the development of lipid nanoparticles for nucleic acid delivery.  2023, 66: 101705.
  2. Mendonça, al. Design of lipid-based nanoparticles for delivery of therapeutic nucleic acids. Drug Discov Today. 2023, 28(3): 103505.
  3. Lu, J. et al.Regulatory perspective for quality evaluation of lipid nanoparticle-based mRNA vaccines in China.  2023, 84: 101700.


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