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The Role of Lipids in Enhancing the Stability of Nanoparticle Formulations

Lipid nanoparticles (LNPs) have shown enormous promise in delivering hydrophobic therapeutics and nucleic acid drugs. Nonetheless, significant challenges remain to enable clinical translation, such as ensuring adequate particle stability for long-term storage.

Lipids as Stabilizers

Lipids are an integral part of any mRNA lipid nanoparticle formulation. They provide a physical and chemical shield from the environment to protect the lipid core from perturbation and degradation, and they also play a crucial role in stabilizing nucleic acid cargos within LNPs.

Incorporating lipids into LNPs is a critical factor in optimizing their transfection efficacy. Lipids’ choice and concentration in the particle dramatically influence the system’s stability, delivery, and endosomal disruption properties. The ionizable lipids used in the construction of LNPs for mRNA and siRNA have pKa values ranging from 9-11, and this low pKa value is critical for favorable electrostatic interactions with the negatively charged backbone of the encapsulated nucleic acid.

As such, incorporating high levels of “helper” lipids (i.e., DSPC, ESM, and cholesterol) into the core of LNPs is expected to enhance their stability and improve their transfection efficiency. This is supported by the observation that LNP mRNA systems containing high (40 mol %) amounts of ESM exhibit comparable or even superior protein expression levels in vitro and in vivo.

Furthermore, cryogenic-transmission electron microscopy characterization of LNPs formulated with DSPC and reduced amounts of ionizable lipid show a bilayer structure consisting of a hydrophobic lipid core composed of triolein (TO) surrounded by a lipid monolayer.


Choosing the optimal lipid is crucial for formulation and determining how long LNPs circulate in the bloodstream. The lipid’s chemical structure and average MW directly influence critical physicochemical properties of the LNP, including size, stability, in vivo distribution, and transfection efficiency.

Lipids with low MWs promote NP self-assembly during fabrication and stabilize the particle once exposed to physiological conditions such as shear forces and glycerol concentrations. On the other hand, longer lipid chains prevent self-assembly and aggregation by creating a steric barrier that impedes interaction with endogenous lipid-associating entities such as lipoproteins and other cysts.

The grafting of PEG chains onto the lipid corona has been shown to increase NP stability in the bloodstream significantly. The high conformational flexibility of the grafted PEG chains creates a hydrated cloud with a large excluded volume that sterically precludes NP interaction with surrounding lipids, blood components, and other NPs. Furthermore, the presence of PEG makes the interpenetration of foreign matter, such as cellular particles and enzymes, thermodynamically unfavorable.

In addition to improving the stability of the lipids, PEG coating has been shown to extend circulation times in the bloodstream. However, determining the optimal PEG molecule for this purpose remains challenging due to its complex molecular structure and many possible combinations of carbon chain lengths and functional groups. NMR spectroscopy has been used to investigate the effects of PEG molecule molar ratios and carbon chain lengths on adsorption, transfection efficiency, and circulation times in the blood.


The main component of cellular membranes is phospholipids. They are asymmetrical molecules with two fatty acid chains and glycerol attached to a phosphate group. Their chemical structure varies depending on the number, type, position, and chain lengths of the fatty acids. Natural phospholipids include lecithin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Synthetic lipids are generally described by the abbreviated name ‘glycerol-phosphatidyl-alcohol’ or ‘glycerol–phosphate–alcohol.’

The phosphate groups of phospholipids are hydrophilic (attracted to water), and the fatty acid tails are hydrophobic (repelled by water). In aqueous solutions, phospholipid molecules will spontaneously assemble into spherical micelle structures. This bilayer formation enables the separation of the fluid inside the cell from the liquid surrounding it.
Liposomes can be used to deliver drugs or nucleic acid cargo. In addition, they can serve as vehicles for transporting cytoplasmic proteins and enzymes as well as signaling molecules. Phospholipids can also be enzymatically cleaved from their glycerol backbone, which releases fatty acid residues to form a phospholipid that can generate energy by oxidation.
Lipids can also be modified to introduce ionizable functional groups, enabling various additional cellular functions. For example, zwitterionic lipids can activate the STING pathway in dendritic cells. Lipid-mRNA nanoparticles with zwitterionic ionizable lipids have been shown to escape the endosome to promote efficient protein expression and genome editing.


Cholesterol is a non-polar lipid found in animal cell membranes and other tissues. It has a unique structure consisting of a sterol backbone with four hydrocarbon rings and a hydroxyl group, which is different from the two other major types of water-insoluble lipids: triacylglycerols (TAG) and phospholipids. It is essential in several biological processes, including cholesterol synthesis (by HMG-CoA reductase), cell signaling, and bile secretion. Cholesterol has also been found to promote nucleic acid encapsulation in LNPs and mediate their endosomal uptake by binding to plasma proteins supporting cellular uptake.
Studies have shown that incorporating cholesterol into LNP formulations significantly enhances their stability, allowing them to circulate in the bloodstream for extended periods.
Using the ethanol-dilution/rapid mixing formulation technique, we have observed that adding increasing amounts of triolein (TO), a non-polar lipid that is insoluble in lipid bilayers, to the DSPC/Chol composition improves the long circulation lifetimes of LNP mRNA systems without affecting entrapment efficiency. These systems exhibit a stable internal solid core surrounded by a lipid monolayer, as visualized by cryogenic transmission electron microscopy.

This is because TO is less dense than DSPC and thus prefers to be incorporated into the interior of the bilayer, where it can form a bleb-like structure alongside deprotonated ionizable lipids. This inversion of the typical bilayer lipid assembly allows TO to effectively stabilize the core region of the LNP and prevent it from aggregating and losing its encapsulated cargo.

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