Encapsulation of mRNA in Therapeutics Like Lipid Nanoparticles Probed by Deep-UV Resonance Raman Spectroscopy

Messenger RNA (mRNA) therapeutics have emerged as a powerful platform for treating a wide range of diseases, but their clinical success depends on overcoming the issues of instability and delivery. mRNAs are typically unstable and require encapsulation in lipid nanoparticles (LNPs) for efficient delivery. This study quantifies free and encapsulated mRNAs within LNPs in a model vaccine using deep-UV resonance Raman (DUVRR) spectroscopy. DUVRR spectroscopy with excitation at 266 nm, matching the maximum UV absorption of mRNA, enables spectral differentiation of mRNA based on its degree of encapsulation─from fully encapsulated to partially exposed to completely free. Raman spectra were acquired from the samples with progressively decreasing lipid concentrations while maintaining a fixed mRNA concentration; vibrational modes associated with mRNA-lipid interactions were identified. Principal component analysis (PCA) revealed spectral variations linked to the lipid presence, especially between 1270 and 1800 cm^-1. Two-trace two-dimensional correlation spectroscopy (2T2D-COS) was employed to extract the 1322 cm^-1 band as a quantitative spectroscopic indicator of the lipid-mRNA interaction. This is the first use of 2T2D-COS for quantitative analysis of complex (bio)chemical systems. The work highlights the unsurpassed potential of DUVRR spectroscopy to analyze mRNAs within LNPs.