Development and Preclinical Evaluation of Virus Like Particle Vaccine Against COVID-19 Infection

In the biomedical field, there has been a requirement for developing theranostic nanomaterials with higher biosafety, leading to both diagnosis and therapy. Methylene blue (MB+) is an organic dye with both photoluminescence (PL) and photosensitization abilities to generate singlet oxygen (1O2). However, MB+ easily loses its generation ability by hydrogen reduction in vivo or by forming aggregates. In this study, MB+ immobilized on biocompatible hydroxyapatite (HA) nanoparticles was applied for the bifunctions of efficient PL and photosensitization. The MB+-immobilized HA nanoparticles (MH) formed aggregates with sizes of 80–100 nm in phosphate buffer (PB). The generation amount and efficiency of 1O2 from the nanoparticles in PB seem to depend on the immobilized MB+ amount and the percentage of the monomer, respectively. Considering the larger immobilized amount and percentage of the MB+ monomer, it was found that there was MH with the lower generation amount and efficiency of 1O2 to exhibit the highest PL intensity. The photofunctional measurement of MB+ revealed the state of MB+ molecules on the HA surface, and it was suggested that the MB+ molecules immobilized on the MH surface would form more hydrogen bonds to change their excitation states. In the cellular experiments, the Hela cancer cells reacted with the nanoparticles and showed red-color PL, indicating cellular imaging. Furthermore, the adherent cell coverage decreased by 1O2 generation, indicating the importance of the immobilization amount of the MB+ monomer. Therefore, theranostic nanomaterials with biosafety were successfully synthesized to show two photofunctions, which provide both cellular imaging and photodynamic therapy by the nanohybrid system between HA and MB+.

Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.

SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.