Nanomaterial-Assisted Stabilization of Lipid Nanoparticles During Lyophilization: Spectroscopic and Microscopic Evidence

The storage durability of nucleic acid treatments based on lipid nanoparticles (LNPs) can be enhanced by lyophilization; however, freeze-drying often causes excipient crystallization, loss of colloidal integrity, and nanoparticle aggregation. While vitrification is provided by conventional molecular cryoprotectants, their ability to fully prevent mechanical and interfacial stresses produced during freezing and drying is remains limited. In this work, we provide spectroscopic and microscopic evidence consistent with improved solid-state and colloidal stability of LNPs after lyophilization. The model system was a commercially available luciferase-encoding mRNA-LNP formulation (LNP-Luc). X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR) analyses indicate altered hydrogen-bonding interactions and increased surface exposure of oxygen-containing functional groups in nanomaterial-assisted formulations. Scanning electron microscopy (SEM) demonstrates the formation of porous and structurally homogeneous amorphous cakes, while transmission electron microscopy (TEM) confirms preservation of nanoparticle morphology following reconstitution. Excipient crystallization suppression is further demonstrated by X-ray powder diffraction (XRPD). Collectively, these findings support nanomaterial-assisted freeze-drying as a promising strategy for developing reliable, cold-chain-independent LNP formulations and provide mechanistic insight into the multiscale stability of LNPs during lyophilization.