Journal of controlled release : official journal of the Controlled Release SocietySep 24, 2026

How protective substances affect the stability of mRNA lipid nanoparticles after freeze-drying and high heat storage

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Sep 25, 2026

Abstract

Lyophilization with a sucrose: trehalose: PVP ratio of 1:1:1 at a 1:20 lipid-to-lyoprotectant ratio maintained stability of mRNA-LNPs after one month at 40°C.

The critical quality attributes (CQAs) of lyophilized mRNA-LNPs were influenced by both the composition of lyoprotectants and the lipid-to-lyoprotectant weight ratio.
A 1:20 lipid-to-lyoprotectant ratio provided the best performance in maintaining particle size and mRNA encapsulation efficiency.
Using sucrose in combination with trehalose and PVP resulted in limited changes in transfection efficiency at the optimal ratio compared to other formulations.
A lipid-to-sucrose ratio of 1:10 stabilized particle size and encapsulation but decreased transfection efficiency relative to the 1:20 ratio.
Lyophilization with sucrose alone led to crystallization at elevated temperatures, which compromised its protective capabilities.

Simplified

Lyophilization has the potential to enhance mRNA-LNP stability at elevated temperatures. The present study aimed to comprehensively evaluate the influence of lyoprotectants on critical quality attributes (CQAs) and function of mRNA-LNPs post-lyophilization and upon storage at 4 °C, 25 °C, and 40 °C. Twelve formulations containing sucrose, trehalose, and polyvinylpyrrolidone (PVP) and their combinations at three total lipid-to-lyoprotectant weight ratios (1:5, 1:10, and 1:20) were studied. The CQAs of lyophilized mRNA-LNPs depended on the lyoprotectant composition and the lipid-to-lyoprotectant weight ratio, with the 1:20 ratio performing best. At this ratio, mRNA-LNPs containing sucrose, sucrose: trehalose 1:1, and sucrose: trehalose: PVP 1:1:1 showed limited change in particle size, mRNA encapsulation efficiency, and in-vitro transfection efficiency compared to other formulations. Interestingly, a lipid-to-sucrose 1:10 ratio provided stabilization of particle size and encapsulation post-lyophilization but resulted in reduced transfection efficiency relative to the 1:20 ratio. Thus, similarities in physicochemical properties did not assure equivalent biological function. After one month of storage at up to 40 °C, lyophilization with sucrose: trehalose: PVP = 1:1:1 at a 1:20 ratio enabled mRNA-LNPs to maintain particle size distribution, encapsulation efficiency, mRNA integrity, and in-vitro transfection. The enhanced stability of the formulation is likely due to its higher glass transition temperature of the lyoprotectant combination. In contrast, at 40 °C, when sucrose was used as the sole lyoprotectant it crystallized, reducing its protective effect. The results highlight a strategy for high-temperature stability of mRNA-LNPs as a reference for formulation development.

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How protective substances affect the stability of mRNA lipid nanoparticles after freeze-drying and high heat storage

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