Trehalose-Loaded Lipid Nanoparticles Enhance mRNA Vaccine Stability and Bridge In Vitro-In Vivo Efficacy Gap

A breakthrough in mRNA vaccine查看药品详情 stability has emerged from researchers who developed an innovative dual-function trehalose查看药品详情 loading strategy that addresses critical challenges in vaccine storage and efficacy. The approach incorporates trehalose both externally as a lyoprotectant and internally within lipid nanoparticles (搜索)搜索该药品 (LNPs), significantly enhancing mRNA vaccine performance during freeze-drying and long-term storage.

The research team prepared trehalose查看药品详情-loaded LNPs (TL-LNPs) using a nanoprecipitation and solvent evaporation method, achieving a trehalose encapsulation efficiency of 17.7 ± 0.9%. The freshly prepared formulations showed similar physicochemical properties, with TL-LNPs exhibiting a size distribution of approximately 154.3 ± 1.3 nm and a zeta potential of around 28 mV, comparable to conventional LNPs.

Enhanced Transfection Efficiency Through Cellular Protection

The TL-LNPs demonstrated remarkable improvements in transfection efficiency compared to conventional formulations. In vitro studies using HEK293T cells revealed that TL-LNPs significantly outperformed both trehalose查看药品详情-mixed LNPs (TM-LNPs) and standard LNPs without trehalose, with statistical significance of p < 0.001 and p < 0.0001, respectively.

The enhanced performance stems from trehalose查看药品详情's ability to protect cells from oxidative stress (搜索)搜索该疾病 induced by cationic LNPs. TL-LNPs transported significantly more trehalose into cells during transfection compared to TM-LNPs (p < 0.01), providing superior cellular protection. The research demonstrated that TL-LNPs significantly reduced cell apoptosis and markedly improved cell viability by decreasing intracellular reactive oxygen species (ROS (搜索)搜索该靶点) levels.

Mechanistic studies revealed that TL-LNPs reduced the percentage of ROS (搜索)搜索该靶点-positive cells and significantly decreased intracellular malondialdehyde查看药品详情 (MDA) levels compared to TM-LNPs (p < 0.01). Additionally, TL-LNPs increased intracellular superoxide dismutase查看药品详情 (SOD) and glutathione查看药品详情 (GSH) levels, indicating enhanced antioxidative defense within cells.

Superior Storage Stability and mRNA Preservation

The freeze-drying optimization process identified -80°C as the optimal pre-freeze temperature, with 20% (w/v) trehalose查看药品详情 concentration providing the best results. After freeze-drying, both FDTM-LNPs and FDTL-LNPs maintained excellent colloidal stability and mRNA encapsulation efficiency, with mRNA integrity remaining largely intact.

During storage at 4°C, FDTL-LNPs demonstrated superior stability compared to conventional formulations. After 4 weeks of storage, FDTL-LNPs retained over 90% of mRNA integrity, while FDTM-LNPs maintained only about 74% integrity. This enhanced chemical stability was attributed to the "hydrogen bond replacement" mechanism, where internal trehalose查看药品详情 forms hydrogen bonds with mRNA, replacing water-mRNA interactions during lyophilization.

Bridging the In Vitro-In Vivo Performance Gap

A critical finding was the ability of TL-LNPs to maintain consistent performance between in vitro and in vivo evaluations. After 4 weeks of storage, FDTL-LNPs showed negligible decrease in in vivo bioluminescence, while FDTM-LNPs exhibited obviously decreased protein expression compared to 2-week storage samples (p < 0.01), retaining only about 76% of fresh LNP luminescent intensity.

The researchers attributed this discrepancy to the biological effects of trehalose查看药品详情 on transfected cells and the differences between in vivo and in vitro environments. The diminished in vivo performance of FDTM-LNPs likely resulted from rapid diffusion of external trehalose, interfering with co-delivery into targeted antigen-presenting cells.

Molecular Mechanisms of Cellular Protection

The study revealed that TL-LNPs significantly reduced cytotoxicity (搜索)搜索该疾病 by decreasing intracellular oxidative stress (搜索)搜索该疾病. Nuclear factor erythroid 2-related factor 2 (Nrf2 (搜索)搜索该靶点) expression analysis showed that cationic LNPs induced significant oxidative stress, with Nrf2 predominantly localized in the nucleus. However, treatment with TM-LNPs or TL-LNPs markedly reduced Nrf2 expression in both nucleus and cytoplasm, indicating reduced oxidative stress and decreased cellular need for Nrf2's protective function.

Comparative studies using vitamin C as a positive control confirmed that antioxidant loading within LNPs effectively enhanced transfection efficiency. Vitamin C-loaded LNPs (VCL-LNPs) significantly outperformed vitamin C-mixed LNPs (VCM-LNPs), supporting the hypothesis that co-delivery of protective agents with mRNA enhances overall performance.

Implications for Vaccine Development

The dual-function trehalose查看药品详情 loading strategy addresses multiple challenges in mRNA vaccine查看药品详情 development simultaneously. By preserving both colloidal stability of LNPs and chemical stability of mRNA while providing cellular protection, this approach offers significant advantages over current lyophilization methods that focus primarily on maintaining structural integrity.

The research demonstrates that this strategy could reduce risks and costs associated with inaccurate efficacy evaluations during storage, particularly important for vaccine distribution in resource-poor countries where maintaining ultracold storage conditions is challenging and expensive.

The findings suggest that the internal trehalose查看药品详情 co-delivery mechanism provides a more reliable approach to maintaining vaccine efficacy during long-term storage compared to conventional external lyoprotectant methods, potentially revolutionizing mRNA vaccine查看药品详情 stability and global accessibility.