NANOSCALE MAPPING OF PACKING DOMAINS IN MODEL LIPID NANOPARTICLE SUPPORTED MEMBRANES VIA TIP-ENHANCED RAMAN SPECTROSCOPY

Lipid nanoparticles (LNPs) are delivery vehicles for nucleic acid-based therapeutics. LNPs are composed of four lipid species: an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid, which self-assemble due to their amphiphilic nature. One of the main unanswered questions regarding their structure is how lipid membrane properties affect transfection: its lipid packing; how closely the lipid molecules are arranged together, may influences membrane hydration,  cellular uptake and cargo release. It is known that lipid mixtures are predisposed to self-organize into coexisting domains, driven by nonideal mixing arising from differential intermolecular interactions among membrane components. These domains, referred to as "nanodomains", are defined by an upper size boundary set by the diffraction limit of light, with an estimated size within the range of 4 to 200 nm. By definition, nanodomains constitute heterogeneities that fall below this resolution threshold and are therefore inaccessible to standard fluorescence microscopy. Approaches for imaging fall short: giant unilamellar vesicle imaging relies on fluorescent probes that perturb lipid packing and bias phase partitioning, cryo-TEM captures only two-dimensional projections of particles, and X-ray scattering yields ensemble-averaged information with no access to local or intraparticle organization. No current traditional technique has been able to image lateral organization of complex lipid membranes at the nanoscale. Tip-enhanced Raman spectroscopy (TERS) has emerged as a tool for label-free, nondestructive chemical characterization at the nanoscale, combining non-contact scanning probe microscopy with confocal Raman spectroscopy. Other techniques provide topographical resolution but lack spectroscopic insight, or instead rely on extrinsic labeling to achieve chemical contrast, TERS overcomes both limitations by coupling subdiffraction spatial resolution with molecular vibrational specificity. Supported Langmuir–Blodgett monolayer membranes have been explored as model lipid membranes due to their defined chemical properties, and have been used extensively to probe phase-separated domains. In this work, using hyperspectral TERS imaging, we demonstrate the existence of discrete packing domains in Langmuir–Blodgett model membranes of LNP  lipid compositions. By employing lipids with distinct crytical packing parameters, we were able to probe nuanced packing orders. Furthermore, we were able to probe subtle spectral variations across the hyperspectral map, demonstrating our capability to resolve nanoscale heterogeneities.