Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency

Abstract

The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA‐rich “bleb” structures. Here, it is shown that such structures can also be induced for LNPs containing nominally less active ionizable lipids by formulating them in the presence of high concentrations of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, with LNP mRNA systems prepared using 300 mm sodium citrate buffer displaying maximum transfection. The improved transfection potencies of LNP mRNA systems displaying bleb structure can be attributed, at least in part, to enhanced integrity of the encapsulated mRNA. It is concluded that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability and that optimization of ionizable lipids to achieve enhanced potency may well lead to improvements in mRNA integrity through formation of the bleb structure rather than enhanced intracellular delivery.

Dual Ethanolamine Head Groups in Ionizable Lipids Facilitate Phospholipid-free Stable Nanoparticle Formulation for Augmented and Safer mRNA Delivery

“…To gain further insights, we employed thioine dye staining to visualize the localization of RNA within LNPs using Cryo-EM 35 . In the case of SM-102 LNP, the RNA density was clearly visible on the periphery of the dense core beneath the outer shell, consistent with prior observations, suggesting that ionizable lipids within SM-102 LNP redistributed into the core when equilibrated in a neutral buffer 24 . Interestingly, LQ-3 iPLX and LNP did not exhibit enhanced contrast due to RNA staining, indicating that LQ-3 forms a tighter complex with RNA, impeding the interaction with thionine dye.…”

“…Remarkably, recent research has unveiled that an increased proportion of phospholipids can enhance mRNA delivery efficacy both in vitro and in vivo , particularly when eff sphingomyelin (ESM) substitutes DSPC 23 . Nonetheless, DSPC tends to favor bilayer structures, resulting in LNP formation with bilayer bleb-like structures under stressful conditions 24 or when elevated DSPC quantities are employed 25 . Replacing DSPC with DOPE circumvents bleb formation, as DOPE prefers a hexagonal H phase, which is incompatible with bilayer structures 26,27 .…”

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue

“…Notably, differences in morphology between individual LNP, including bleb formation has recently been attributed to occur during buffer exchange. [ 18 ] Interestingly, in the context of mRNA delivery, such bleb formation has been a found to improve transfection efficiency.…”

mRNA Cancer Vaccines: Construction and Boosting Strategies

“…For example, Cheng et al demonstrated that high concentrations of mRNA/ LNP prepared by sodium citrate buffer (300 mM) exhibited the optimal mRNA transfection efficiency. 99 2.3.2. Polymer-Based Vectors.…”

“…Moreover, optimizing the buffer composition in the LNP preparation process is also beneficial for improving the mRNA transfection efficiency. For example, Cheng et al demonstrated that high concentrations of mRNA/LNP prepared by sodium citrate buffer (300 mM) exhibited the optimal mRNA transfection efficiency …”