Methodology

The researchers utilized in situ small-angle X-ray scattering (SAXS) during the microfluidic mixing process and subsequent dialysis to track the structural evolution of LNPs, a technique used to analyze the size, shape, and internal structure of particles in a sample by measuring the scattering of X-rays at very small angles. Additionally, they employed dynamic light scattering (DLS), small-angle neutron scattering (SANS), and cryo-transmission electron microscopy (cryo-TEM) to characterize the final formulations. The study focused on a benchmark lipid composition and examined the effects of incorporating different nucleic acids, including calf thymus DNA, polyadenylic acid (polyA), and polyuridylic acid (polyU).

Key Results and Findings

Structural Evolution during Formulation

The structure of LNPs evolves significantly during the microfluidic mixing process, but the fully developed structure only emerges during the dialysis stage. Initial structural changes, including the formation of ‘protoLNPs’, were observed as early as during the mixing stage, driven by the self-assembly of lipids in response to the mixing environment.

Impact of Nucleic Acid Cargo

The type of nucleic acid cargo significantly influences the colloidal stability of the LNPs. For instance, LNPs incorporating polyU exhibited extensive aggregation, leading to decreased stability, whereas those containing DNA or polyA showed greater stability. The degree of base-pairing in the nucleic acids was identified as a key factor affecting stability, with polyU, which is primarily single-stranded, leading to more aggregation compared to the double-stranded DNA or the partially paired polyA.

Final Structure and Stability

The study revealed that while the internal structure of LNPs is sensitive to the type of nucleic acid cargo, the overall lipid composition of the nanoparticles remains relatively consistent. The research highlighted that LNPs containing different nucleic acids coexisted with empty LNPs (those without nucleic acids), and the proportion of these populations varied with the nucleic acid-to-lipid ratio (N:P ratio). Notably, the final LNP formulations contained distinct populations of particles with and without nucleic acids, which could be differentiated based on their internal structure as observed in SAXS data.

Influence of N:P Ratio

The study also found that the N:P ratio influenced the internal structure of the LNPs. A higher N:P ratio, which corresponds to a lower nucleic acid loading, resulted in a decrease in the size of the LNPs and altered the internal lipid composition, particularly increasing the proportion of MC3 in the core.

Implications and Future Directions

The findings from this study provide crucial insights into the formulation of LNPs, highlighting the importance of understanding the structural dynamics during the production process. The demonstrated sensitivity of LNP structure to the type of nucleic acid cargo and the formulation conditions suggests that tailored approaches are necessary for optimizing LNPs for different therapeutic applications. The observation of distinct populations of LNPs with and without nucleic acids underscores the need for further research into the factors that control cargo loading efficiency and distribution.

This study also emphasizes the potential of advanced scattering techniques like SAXS and SANS in providing detailed structural information that can guide the rational design of more effective LNP formulations. Future research could explore the scalability of these findings and their implications for large-scale manufacturing of LNPs, particularly in the context of therapeutic mRNA delivery. One approach would be to develop high-throughput microfluidic systems that can produce LNPs with consistent structural integrity on an industrial scale, and by optimizing tangential flow filtration (TFF) processes to maintain the stability and efficacy of mRNA-LNP formulations during large-scale production.

Reference

The research was conducted by Jennifer Gilbert, Federica Sebastiani, Marianna Yanez Arteta, Ann Terry, Anna Fornell, Robert Russell, Najet Mahmoudi, and Tommy Nylander, with significant contributions from Lund University and AstraZeneca. The full study is available in Journal of Colloid and Interface Science (2024), DOI : 10.1016/j.jcis.2023.12.165.