Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles

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.

Nucleic acid degradation as barrier to gene delivery: a guide to understand and overcome nuclease activity

“…330,331 Although LNPs can shield mRNA from chemical degradation by nucleases, it has been suggested that water present in the core of mRNA LNPs results in non-enzymatic mRNA hydrolysis, which is now considered as the main mechanism for mRNA degradation and the limited storage stability of mRNA LNPs. 317,332 To prevent hydrolysis, the currently approved Comirnaty should be kept at À80 1C, having a large impact on the distribution and storage. Freeze drying of mRNA LNPs has been explored to enable storage at higher temperature, where both the type of cryoprotectants and the buffers used greatly impact the stability of mRNA LNPs.…”

“…330,331 Although LNPs can shield mRNA from chemical degradation by nucleases, it has been suggested that water present in the core of mRNA LNPs results in non-enzymatic mRNA hydrolysis, which is now considered as the main mechanism for mRNA degradation and the limited storage stability of mRNA LNPs. 317,332…”

Next-generation materials for RNA–lipid nanoparticles: lyophilization and targeted transfection

“…1). 2,[8][9][10][11][12][13][14][15][16][17][18][19][20] The ionizable lipid SM-102 (Lipid H) is utilized in the mRNA-1273 (Spikevax) vaccine, and the ionizable lipid ALC-0315 is used in the BNT162b2 (Comirnaty) vaccine (Fig. 1).…”

“…2 Most research on mRNA-LNPs, miRNA-LNPs or siRNA-LNPs has been devoted to pursuing highly efficient RNA expression in vitro and in vivo. [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] Only a few studies have investigated the long-term storage of RNA-LNPs. 1,[23][24][25] One of the most efficient methods to store RNA-LNPs for a long time is freeze-drying (lyophilization).…”

mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles

“…There are a limited number of published studies on freezing iLNPs. Sugar‐based cryoprotectants, such as sucrose, mannitol and trehalose have been reported, and the selection of buffers and freezing conditions have been investigated (Ball et al., 2017; Henderson et al, 2022; Kafetzis et al., 2023; Kim et al., 2023; Zhao et al., 2020). The optimal conditions reported for freezing mRNA‐iLNPs appears to be variable, depending on the ionizable lipid or lipidoid and RNA cargo.…”