Characterization of mRNA Lipid Nanoparticles by Electron Density Mapping Reconstruction: X-ray Scattering with...

Area of Science:

• Biophysics
• Nanotechnology
• Materials Science

Background:

• Messenger RNA-lipid nanoparticles (mRNA-LNPs) are crucial for vaccine and therapeutic delivery.
• Characterizing the internal structure of mRNA-LNPs is essential for optimizing their efficacy and stability.
• Existing methods may have limitations in resolving the detailed internal architecture of mRNA-LNPs.

Purpose of the Study:

• To evaluate the feasibility of using Small Angle X-ray Scattering (SAXS) combined with the Density from Solution Scattering (DENSS) algorithm for characterizing mRNA-LNP internal architecture.
• To develop a 3D model of average individual mRNA-LNPs using the DENSS algorithm.
• To validate the SAXS-DENSS approach against established techniques like cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS).

Main Methods:

• Small Angle X-ray Scattering (SAXS) was employed to collect scattering data from mRNA-LNPs.
• The Density from Solution Scattering (DENSS) algorithm was utilized to reconstruct a 3D electron density map of the mRNA-LNPs.
• Cryogenic transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) were used for cross-validation of size, morphology, and internal structure.

Main Results:

• SAXS, cryo-TEM, and DLS confirmed a core-shell structure with an mRNA-rich core and surrounding lipids.
• The DENSS algorithm successfully generated 3D models distinguishing between mRNA and lipid components based on electron density.
• Accurate modeling of mRNA-LNP morphology, including ellipsoidal shapes with 'bleb' or two-compartment structures, was achieved.
• SAXS detected structural changes, such as increased radius of gyration indicating mRNA leakage, after three freeze-thaw cycles.

Conclusions:

• The SAXS-DENSS approach provides a powerful tool for physicochemical characterization of mRNA-LNPs.
• This method enables detailed analysis of internal structure and stability, aiding in mRNA-LNP development.
• The technique facilitates the study of structural changes relevant to LNP formulation and storage conditions.