Journal of nanobiotechnologyApr 11, 2026

How Ionizable Lipid Amounts in mRNA Lipid Nanoparticles Affect mRNA Release and Protein Production Inside Cells

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Updated Apr 12, 2026

Abstract

Lipid nanoparticles containing lower levels of ionizable lipids (~30 mol%) achieved significantly higher in vitro transfection efficiency and in vivo mRNA expression.

Optimized lipid nanoparticles demonstrated enhanced intracellular mRNA release compared to standard formulations.
Similar cellular uptake and endosomal escape were observed across different lipid compositions.
Intracellular mRNA-LNP dissociation is identified as a crucial factor affecting translational efficiency.
Fluorescence analyses confirmed greater cytosolic release of mRNA from lipid nanoparticles with reduced ionizable lipid content.
Formulations with lower ionizable lipid levels elicited stronger immune responses in mice.

Simplified

Messenger RNA (mRNA) therapeutics rely on lipid nanoparticles (LNPs) for effective intracellular delivery, yet the intracellular release of mRNA from these carriers remains an underexplored barrier to translation. In this study, we investigate how the ionizable lipids within clinically relevant LNP formulations influence mRNA delivery and translation. By systematically varying the molar ratio of ionizable lipids (SM-102 and ALC-0315) while maintaining other lipid components constant, we found that LNPs containing lower ionizable lipid levels (~ 30 mol%) achieved significantly higher in vitro transfection efficiency and in vivo mRNA expression compared to standard clinical formulations (50 mol% for Moderna; 46.3 mol% for Pfizer/BioNTech). Notably, this enhancement occurred despite similar cellular uptake and endosomal escape, implicating intracellular mRNA-LNP dissociation as a previously underappreciated determinant of translational efficiency. Fluorescence colocalization analyses confirmed greater cytosolic mRNA release from LNPs with reduced ionizable lipid content. Moreover, these optimized formulations elicited stronger antigen-specific humoral and cellular immune responses in mice, highlighting their potential for improved mRNA performance. Collectively, these findings identify intracellular mRNA dissociation-rather than endosomal escape alone-as a key bottleneck in LNP-mediated delivery and demonstrate that fine-tuning ionizable lipid composition can substantially enhance mRNA performance.

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Competing interests

Declarations. Ethics approval and consent to participate: All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Guidelines of the Institutional Animal Care and Ethics Committee of the University of Massachusetts Lowell and University of Houston. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

PubMed

How Ionizable Lipid Amounts in mRNA Lipid Nanoparticles Affect mRNA Release and Protein Production Inside Cells

Abstract

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