Trehalose-loaded LNPs enhance mRNA stability and bridge in vitro in vivo efficacy gap

Abstract

Lyophilization enhances mRNA vaccine stability, but conventional approaches using external trehalose for lipid nanoparticle (LNP) colloidal stability neglect mRNA chemical degradation and are compromised in vivo efficacy. Here, we report a dual-function trehalose strategy integrating its external and internal roles within LNP. This strategy enables trehalose to externally form a vitrified matrix that preserves LNP colloidal integrity, while internally stabilizing mRNA through hydrogen bonding, markedly reducing chemical degradation during storage compared to LNP relying solely on externally added trehalose. Crucially, co-loaded trehalose is co-delivered into cells, bridging the in vitro–in vivo gap by mitigating oxidative stress through reduced reactive oxygen species (ROS) and malondialdehyde (MDA) alongside elevated glutathione (GSH) and superoxide dismutase (SOD). This is corroborated by downregulated cytoplasmic and nuclear nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Our strategy provides a simple, universally adaptable, and scalable method to enhance mRNA-LNP formulations stability without exogenous components or complex lyophilization steps.

Introduction

Vaccines are one of the most cost-effective methods to prevent infectious diseases. mRNA vaccines are promising candidates due to their rapid development and low-cost manufacture1. Implementing a relatively simple, independent manufacturing process significantly expedited both time and cost in the development of a new vaccine, particularly amidst a pandemic scenario marked by the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants2. Although mRNA vaccines hold great promise, their clinical translation and commercialization are still encountering challenges, as mRNA is highly susceptible to hydrolysis, oxidation3 and RNase enzymes4. The short shelf life and ultracold storage requirement of mRNA vaccines slow down their distribution mostly in resource-poor countries of the world, as maintaining ultracold storage conditions is expensive and difficult to arrange5,6. Freeze-drying is a commonly used technique to improve the stability of mRNA vaccines7,8. Current typical freeze-drying methods relying on simply mixing lyoprotectants (e.g., trehalose or sucrose) outside the formulation9,10 and optimizing the lyophilization process, including parameters such as cycle times, temperature, vacuum levels, and buffer conditions. While these factors are critical, they are costly and require precise control, often necessitating expensive equipment and complex operations, which lead to significant batch-to-batch variations and increased production costs. Moreover, these methods mainly focus on maintaining the colloidal stability of mRNA-loaded lipid nanoparticles (LNPs)11. Nevertheless, while some formulations retain structural integrity and encapsulation efficiency (EE) after lyophilization, their in vivo transfection efficiency remains reduced for unknown reasons8,10,12. Therefore, it is essential to develop new strategies to enhance the stability of lyophilized mRNA vaccines, bridge the in vitro-in vivo stability gap, and reduce the risks and costs associated with inaccurate efficacy evaluations during storage.

The stability or the efficacy of lyophilized mRNA vaccines is mainly determined by: (1) the colloidal stability of the delivery system (e.g., LNPs) (2) the chemical stability of the mRNA molecular, and (3) the effect of lyoprotectants on the targeted cells being transfected. Firstly, lyoprotectants can form a stable glassy state, which immobilizes the formulation within a rigid, amorphous, glassy sugar matrix during freeze-drying. This “vitrification theory” effect significantly decreases the formation of ice crystals and prevent their mechanical damage to the colloidal stability of the delivery system13. Secondly, lyoprotectants form hydrogen bonds with the mRNA, effectively replacing hydrogen bonds that would otherwise form between water and the mRNA during lyophilization. This “hydrogen bonds replacement” helps maintain the native conformation and the chemical stability of mRNA9,14. On the other hand, some lyoprotectants also possess antioxidant properties, offering additional protection to the transfected cells, which are vulnerable to oxidative stress induced by the cationic LNPs15,16. For example, trehalose, which accumulates notably during heat shock and stationary phase across various organisms, can enhance thermotolerance, reduce denatured protein aggregation, and protect cells from oxygen radicals17,18,19. Although lyoprotectants can protect mRNA vaccines through various mechanisms, current external incorporation methods can only maintain the vaccine’s colloidal stability13. The chemical stability of mRNA molecules is often overlooked. Additionally, the immunological effect of lyoprotectants has received little attention, even though they are co-administered with the rehydrated vaccine in humans10,20. It has been reported that trehalose can enhance transfection efficiency when co-existed with transfection reagents16. However, the current lyophilization strategy, which places the lyoprotectant like trehalose externally in LNPs, faces the issue of trehalose being inefficiently co-delivered with the LNPs to target cells in vivo. This may be the reason for the discrepancy between the in vitro and in vivo performance of mRNA efficacy.

To enhance the freeze-drying stability of mRNA-LNP formulations and bridge the gap between the in vitro and in vivo performance, we proposed a dual-function trehalose loading strategy. In this method, trehalose functions not only as an external lyoprotectant but also as a component that is co-loaded with mRNA within LNPs. During freeze-drying, external trehalose protects the colloidal stability of the LNPs, while internal trehalose replaces water and interacts with mRNA via hydrogen bonding, enhancing the chemical stability of mRNA by restricting its molecular mobility. Furthermore, internal trehalose can enter target cells along with LNPs during in vivo evaluation, providing additional protection to transfected cells vulnerable to oxidative stress induced by LNPs and enhancing in vivo transfection efficiency (Fig. 1). With this dual-function strategy, both the chemical stability of the mRNA and the immunological effects of trehalose on targeted cells could be well addressed. Herein, the preparation and characterization of trehalose-loaded LNPs were systematically analyzed. Both the in vitro and in vivo transfection efficiencies of these formulations after storage for various time periods were evaluated. After freeze-drying and storage, the colloidal stability of the formulation and the chemical stability of mRNA were also analyzed.

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Materials

Cationic lipid SM-102, 1,2-distearoyl-sn-glycero-3-PC (DSPC) and cholesterol were purchased from Rhawn (China). Polyethylene glycoldimyristoyl glycerol (PEG2000-DMG) was brought from Shanghai Macklin Biochemical Technology Co., Ltd. Tetrahydrofuran (THF), Bull serum albumin (BSA), Tween-20 , and trehalose were purchased from Sigma-Aldrich. Trypsin-EDTA (0.25%, w/v), fetal bovine serum (FBS), and 1% penicillin/streptomycin were bought from Gibco (CA, USA). EZ Cap^TM Firefly luciferase mRNA was purchased from Apexbio. Firefly Glo Luciferase Reporter Gene Assay Kit was obtained from Yeasen (Shanghai, China). Phosphate-buffered saline (PBS), DMEM medium were obtained from Hyclone Lab (UT, USA). RNase-free water and TPCK-trypsin were purchased from Thermo Fisher Scientific (UK). Trehalose Assay Kit was purchased from Nuominkeda (Wuhan, China) Biotechnology Co., Ltd. RiboGreen assay kit was purchased from Thermo Fisher. CCK8, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) assay kits were purchased from Elabscience. RIPA buffer was purchased from Solarbio (Beijing, China). Reactive Oxygen Species (ROS) detection kit, Nuclear and Cytoplasmic Protein Extraction Kit, and Annexin V-FITC/PI apoptosis detection kit were obtained from Beyotime. Goat Anti-Rabbit IgG H&L polyclonal antibody FITC (PTB96441) and Anti-NFE2L2 polyclonal antibody were purchased from Antibody System (France).

Liu, XH., Song, HP., Tao, LL. et al. Trehalose-loaded LNPs enhance mRNA stability and bridge in vitro in vivo efficacy gap. npj Vaccines 10, 201 (2025). https://doi.org/10.1038/s41541-025-01253-3