(T0930-01-01) Lipid Nanoparticle Morphology and Phase Transitions Investigated through Small Angle X-Ray Scattering and Contrast Variation in a Laboratory Instrument

Purpose: Lipid nanoparticles (LNPs) have successfully been used to deliver RNA as both vaccines and therapeutics. However, the stability and performance of LNPs are highly dependent on their structural properties requiring extensive characterization. Small angle x-ray scattering (SAXS) has emerged an as effective tool to characterize lipid nanoparticle formulations. SAXS can simultaneously determine the size, shape, and internal structure of LNPs in solution. Furthermore, contrast variation SAXS (CV-SAXS) can be used to provide additional details of the LNP morphology of lipid nanoparticles.

Methods: In this study, we investigated the structural properties of RNA loaded using high throughput SAXS measurements with a laboratory instrument (Xenocs Xeuss 3.0). First, we perform CV-SAXS measurements to accurately determine the morphology and scattering length density of the lipid nanoparticles. We also performed measurements in the temperature between 10C-60C and pH values between 4-7.5. We then used a structural correlation map to develop an empirical phase diagram of the LNP formulation.

Results: Using the contrast variation SAXS data we were able to determine that the lipid nanoparticle had a core-shell sphere structure with an enriched outer layer and low-density lipid core. The scattering length density of the core was consistent with the scattering length density of the ionizable lipid- indicating low RNA content. Using the pH-temperature measurements determine an empirical phase diagram for our lipid nanoparticle formulation. With the SAXS data we created a structural correlation map which when clustered revealed 6 structurally similar regions in the phase space. Interestingly we see a strong phase transition at the pKa of our ionizable lipid that shifts to lower pH at higher temperatures. We also noted that the phase transition affected both the overall LNP structure as well as the internal lipid structure.

Conclusion: Contrast variation SAXS was used to unambiguously elucidate the morphology of the LNP. We suggest that similar methods can be used to distinguish between spherical and bleb morphologies. Furthermore, we suggest the method can be used to provide enhanced sensitivity to the RNA loading and structure of the LNP. Using the high-throughput SAXS to measure the LNPs at several pH and temperature values allowed us to create an empirical phase diagram for our formulation. The empirical phase diagram can then serve as a roadmap to understanding the behavior of our LNP in different conditions. Additionally, information about each phase can be obtained through SAXS.