Mesoscopic Structure of Lipid Nanoparticle Formulations for mRNA Drug Delivery: Comirnaty and Drug-Free Dispersions

Lipid nanoparticles (LNPs) have emerged as a versatile platform for mRNA delivery across a range of applications, including disease prevention, cancer immunotherapy, and gene editing. Structural models of mRNA-containing lipid nanoparticles (mRNA-LNPs) have also been proposed based on characterization of samples by using various advanced techniques. Among these, small angle neutron scattering (SANS) has proven essential for elucidating the lipid distribution within mRNA-LNPs, a factor crucial to both their preparation and efficacy. However, recent findings suggest that the mRNA-LNP samples prepared via commercial microfluidic techniques may contain a substantial fraction of drug-free LNPs, casting doubt on the validity of earlier structural models. In this study, we employed contrast variation SANS to characterize both drug-free LNPs and our mRNA-LNP sample, and quantified the proportion of drug-free LNPs present to be ~30% in our mRNA-LNP sample using nano flow cytometry. By removing the contributions of drug-free LNPs from the SANS data of our mRNA-LNP sample, we were able to precisely characterize the structure of mRNA-LNPs. Consequently, we proposed structural models for both drug-free LNPs and mRNA-LNPs. Notably, our analysis revealed similar lipid distributions and shell thicknesses between the two particle types, while the solvent content in mRNA-LNPs was significantly higher, leading to a larger core size. This work not only offers a method for accurately characterizing the structure of mRNA-LNPs, but also establishes criteria for selecting appropriate analytical techniques based on the structural parameters of interest. Therefore, our findings hold significant implications for the mechanistic understanding and quality control of mRNA-based vaccines.

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Mechanistic Insight into pH-Driven Phase Transition of Lipid Nanoparticles

Trollmann,

Böckmann

2024

Preprint

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Lipid nanoparticles (LNPs) are essential delivery vehicles in mRNA-based vaccines, with their functionality largely governed by aminolipids. At low pH, protonated aminolipids facilitate efficient mRNA encapsulation, while their deprotonation at physiological pH drives LNP remodeling into a stable spherical structure, potentially aiding mRNA release during endosomal re-protonation and concomitant restructuring at low pH. Our study uncovers the pH-dependent phase transition of the Comirnaty lipid formulation at the molecular scale, highlighting the intricate interplay between lipid reorganization and aminolipid protonation. We observe a substantial shift in the aminolipid pKa, from 7-8 at the LNP surface to below 4 within the hydrophobic LNP core, resulting in an overall apparent pKa of the lipid nanoparticle of 6.3. The pH-dependent protonation significantly affects the aminolipid's localization, with protonated aminolipids enriched in vicinity of the mRNA and at the LNP surface at neutral pH. The lipid monolayer covering LNPs at neutral pH comprises cholesterol (60-65%), helper phospholipids (20-25%), PEGylated lipids (5%), and a mix of protonated and deprotonated aminolipids (5-10%). These findings provide mechanistic insights into aminolipids behavior in LNPs, offering a foundation for optimizing LNP design to enhance stability, tunable pH responses, and therapeutic efficacy.

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Mesoscopic Structure of Lipid Nanoparticle Formulations for mRNA Drug Delivery: Comirnaty and Drug-Free Dispersions

Cited by 3 publications

References 109 publications

A brief overview of quality by design approach for developing pharmaceutical liposomes as nano-sized parenteral drug delivery systems

A brief overview of quality by design approach for developing pharmaceutical liposomes as nano-sized parenteral drug delivery systems

Structural Characterization of mRNA Lipid Nanoparticles in the Presence of Intrinsic Drug-free Lipid Nanoparticles

Mechanistic Insight into pH-Driven Phase Transition of Lipid Nanoparticles

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