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Physicochemical Stability

Lipid nanoparticle formulations should be both physically stable (nanoparticles should maintain a homogeneous size distribution and a constant level of drug encapsulation), and chemically stable (lipids and APIs should not degrade during storage).

Physical Stability

Fig. 1. Structure of lipid-based drug delivery systems. (Fan, 2021) Fig. 1. Structure of lipid-based drug
delivery systems. (Fan, 2021)

Small lipid nanoparticles with dynamic lipid bilayers could fuse into larger vesicles during storage. Along with or following lipid fusion, these particles can also aggregate, inducing phase separation and leakage of the encapsulated API. Liposomal fusion has been investigated mainly by differential scanning calorimetry (DSC) and fluorescence-based lipid mixing assays.

In the DSC approach, the fusion of two populations of liposomes composed of two types of lipids with distinctive transition temperatures, would introduce a new endothermic phase transition peak upon lipid mixing. Although DSC can achieve label-free analysis of liposomal fusion, this approach only applies to pure lipid systems with pre-known transition temperatures and requires large amounts of liposome samples due to relatively low sensitivity.

In contrast, fluorescence-based strategies, generally based on quenching/dequenching of the Förster resonance energy transfer (FRET) of fluorophore pairs, can achieve sensitive and kinetic measurements, simultaneous visualization, and quantification of the fusion process. Specifically, an aqueous-soluble fluorophore and its counterpart fluorescence enhancer or quencher can be separately encapsulated in the liposome core. Liposomal fusion would then be indicated by changes of the fluorescence intensity upon the complexation of the reagents.

Aggregation of liposomes can be visualized by microscope techniques and quantified by ultraviolet (UV) vis spectroscopy or dynamic light scattering (DLS). Turbidity assays that measure absorbance at 400-600 nm have been commonly used as a facile method to monitor aggregation kinetics of liposomes. Particle size distributions measured by DLS have been used to investigate potential mechanisms of destabilization. Researchers have compared liposomes incorporated with varying amounts of cholesterol and found 30 mol% of cholesterol produced the most stable formulations, as determined by AFM, DLS, and FTIR. Cholesterol can stabilize lipid layers by promoting the cohesion and liquid-ordered phases of lipids.

Chemical stability

The encapsulated API can be extracted and assessed for their chemical stability usually by using LC-MS or identified for their physical states in situ using small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The stability of lipid bilayers and lipid nanoparticle formulations can be impaired by lipid degradation, mainly via hydrolysis and/or oxidation. Lipid degradation rates could be affected by lipid compositions, storage temperatures, buffers, and pH. The original lipid species and their hydrolyzed derivatives, such as fatty acids and lysophospholipids, can be separated and detected by several chromatography approaches as described in the Identification and quantification of lipid species section. For instance, RP-HPLC coupled with evaporative light scattering detector (ELSD) has been used to simultaneously separate and quantify 16 lipoid components including cholesterol, phosphatidylcholine (PC), phosphatidylglycerol (PG), and their degradants in liposomal formulations. Normal phase LC using silica or hydrophilic interaction columns coupled with charged aerosol detector (CAD) has also been used to quantify phospholipid degradants. In addition, LC-MS could be used to further increase the detection sensitivity of the degraded products. Under acidic conditions, the rate of lipid hydrolysis was found to follow the rank order phosphatidylethanolamine (PE) < PC < PG, and was significantly affected by the incorporation of charged molecules in the lipid bilayer.

Services at Creative Biolabs

The physicochemical stability of lipid nanoparticle-formulated drugs influences every aspect of the drug efficiency. Creative Biolabs is a professional provider of the custom delivery vehicle for mRNA. With years of experience accumulated from practice, we are confident in satisfying every custom's requirements. Our platform is equipped with advanced facilities, up-to-date technologies, and a Ph.D. level experts team. All these advantages are the powerful guarantees of our services. If you are interested in our services or you need any assistance with physicochemical stability characterization, please don't hesitate to contact us for more information.


  1. Fan, Y.; et al. Analytical characterization of liposomes and other lipid nanoparticles for drug delivery. J Pharm Biomed Anal. 2021, 192: 113642.
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