Amorphous trehalose as a superior lyoprotectant for high-temperature long-term storage of freeze-dried mRNA-lipid nanoparticles

The large-scale global application of messenger ribonucleic acid–lipid nanoparticles (mRNA-LNPs) is severely restricted by their mandatory ultra-low-temperature storage requirement, which poses critical logistics and transportation bottlenecks, especially in resource-limited regions. Lyophilization serves as a mild and well-established strategy to improve the thermal stability of biotherapeutics; nevertheless, complex physicochemical stresses during the lyophilization process may disrupt the structural integrity of mRNA and destroy the colloidal stability of LNP systems.

Sucrose has been widely recognized as an efficient lyoprotectant, whereas the underlying mechanisms responsible for the stabilizing/destabilizing effects of crystalline and amorphous excipients remain incompletely understood. In this work, differential scanning calorimetry (DSC) and X-ray diffraction (XRD) were utilized to characterize lyophilized samples with diverse formulations. Combined with stability evaluation at 4 °C, 25 °C and 40 °C, the correlations between excipient crystallization behavior, thermodynamic properties and storage stability were systematically clarified. The results revealed that trehalose, with an intrinsically higher glass transition temperature in the amorphous state, exhibits superior lyoprotective capacity compared with sucrose under extreme storage conditions (40 °C), making it a more promising candidate for mRNA-LNP lyophilization. As a typical crystalline excipient, mannitol is inappropriate for single-component lyoprotectant application, as it severely impairs the long-term storage stability of mRNA-LNPs by damaging nanoparticle morphology and reducing nucleic acid encapsulation efficiency.

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Materials

eGFP mRNA gene was synthesized by Yinno Biological Co., Ltd (Shenzhen, China). PolyA tail was synthesized by Roche Life Science (Shanghai, China). 1,2-Dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), Amino cationic lipid SM102, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), High Purity Cholesterol was sourced from AVT Pharmaceutical Co., Ltd (Shanghai, China). Nuclease-Free Water and 100× penicillin-streptomycin solution was acquired from Solarbio Life Sciences.

Han Gao, Jun Ouyang, Chaoyang Du, Qian Shao, Wei-Jie Fang, Amorphous trehalose as a superior lyoprotectant for high-temperature long-term storage of freeze-dried mRNA-lipid nanoparticles, International Journal of Pharmaceutics, Volume 705, 2026, 127546, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.127546.