Lipid nanoparticles own a remarkable potential in nanomedicine, only partially disclosed. While the clinical use of liposomes and cationic lipid-nucleic acid complexes is well-established, liquid lipid nanoparticles (nanoemulsions), solid lipid nanoparticles, and nanostructured lipid carriers have even greater possibilities. However, they face obstacles in being used in clinics due to a lack of understanding about the molecular mechanisms controlling their drug loading and release, interactions with the biological environment (such as the protein corona), and shelf-life stability. To create effective drug delivery carriers and successfully translate bench research to clinical settings, it is crucial to have a thorough understanding of the internal structure of lipid nanoparticles. Through synchrotron small-angle X-ray scattering experiments, we determined the spatial distribution and internal structure of the nanoparticles' lipid, surfactant, and the bound water in them. The nanoparticles themselves have a barrel-like shape that consists of coplanar lipid platelets (specifically cetyl palmitate) that are covered by loosely spaced polysorbate 80 surfactant molecules, whose polar heads retain a large amount of bound water. To reduce the interface cost of bound water with unbound water without stacking, the platelets collapse onto each other. This internal structure challenges the classical core-shell model typically used to describe solid lipid nanoparticles and could play a significant role in drug loading and release, biological fluid interaction, and nanoparticle stability, making our findings valuable for the rational design of lipid-based nanoparticles.

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1’ and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.