Abstract
Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers─HEPES, Tris, and phosphate-buffered saline─with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze-thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.
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