Effect of Formulation Buffer Type on LNP Size

Introduction

Lipid nanoparticle (LNP) fabrication typically starts with rapid mixing of lipids in ethanol and nucleic acid-based cargos prepared in an acidic aqueous buffer solution (buffer pH < pKa of the ionizable lipid). The acidic aqueous buffer is of importance to (1) protonate the ionizable lipids so that they may bind electrostatically with the negatively charged nucleic acid-based cargos and (2) increase the solvent polarity to drive the particle formation and cargo encapsulation. Aqueous buffer solution parameters such as type of buffering species, pH and salt concentration (ionic strength) not only impact cargo stability but also LNP size and size distribution.

Many studies have reported the importance of LNP size on the in vitro/in vivo efficacy. The optimal LNP size largely depends on the intended indication, route of administration and animal species. For example, the ideal particle size for hepatic delivery of intravenously administered LNPs in nonhuman primates (NHPs) is between 50 to 60 nm, while 70 to 80 nm works best in rodents. When LNPs are used as a vaccine and administered by intramuscular injection, different particles sizes ranging from 60-150 nm produced similar immune response in NHPs, but in mice particles > 80 nm performed the best. Therefore, precise size control of LNPs during fabrication is an essential step towards maximizing the desired therapeutic effects.

The LNP size can be tuned in many ways. Increasing the molar ratio of polyethylene glycol (PEG) lipid is commonly employed to reduce particle size, stabilize LNPs and increase their circulation time. However, too much PEG on the particle surface will inhibit the cellular uptake of LNPs. In addition, PEG associated immunogenicity may result in reduced treatment efficacy or unwanted side effects. LNP size can also be adjusted by changing the mixing conditions such as the total flow rate, cargo/lipid solution flow rate ratio, and inline dilution ratio. In this study, we aim to tune the LNP size using different cargo buffer solutions while maintaining the same lipid composition and mixing conditions across all formulations.

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