Biofunctionalization of Micro/Nanoparticles for Neutralizing SARS-CoV-2 Surrogate and Targeting ACE2-expressing Lung Cells

Abstract

The SARS-CoV-2 virus, which led to the COVID-19 pandemic, first emerged in China in the end of 2019 and swiftly propagated to affect the entire world. COVID-19 has caused millions of illnesses and deaths worldwide. The illness from virus infection may lead to acute respiratory distress syndrome (ARDS), necessitating hospitalization in intensive care units and the use of mechanical ventilators for some individuals. Efforts to mitigate the effects of the pandemic have depended heavily on measures like vaccination drives and public health strategies such as the use of masks and maintaining physical distance to curb the virus's transmission. The emergence of SARS-CoV-2 variants has raised concerns due to their increased ability to spread, their potential to lead to more severe illness, and their reduced responsiveness to existing treatments and vaccines. Preventing viral nanoparticles from entering susceptible lung cells is an approach to curb the transmission of SARS-CoV-2 and any future related coronaviruses. In this study, two strategies were harnessed to achieve the goal – (i) blocking SARS- CoV-2 spike proteins on the outer surface of virions, and (ii) blocking recombinant human angiotensin- converting enzyme 2 (hACE2) receptors on the targeting lung cells. Micro/nanoparticles (biotinylated fluorescent polystyrene particles and perfluorooctyl bromide (PFOB)-based oxygen nanoemulsions) tethered with the recombinant hACE2 were designed and fabricated for the neutralization of spike protein pseudotyped lentivirus (employed as SARS-CoV-2 surrogate). For blocking the hACE2 receptors on the target cells, biotinylated fluorescent polystyrene particles tethered with the recombinant receptor-binding domain (RBD) to saturate hACE2 receptors, thereby preventing the SARS-CoV-2 surrogate from infecting cells. Furthermore, since mesenchymal stem cell-derived extracellular vesicles (MSC-derived EVs) have been proven to possess therapeutic potential for repairing lung injuries, an innovative method for the separation of MSC-derived EVs was developed using chitin magnetic microparticles bound with chitin-binding domain (CBD) fusion with self- cleaving intein tag and lactadherin C1C2, which has a high affinity to phosphatidylserine (PS) exposed on EV membranes. As a result, purified MSC-derived EVs tethered with RBD of SARS-CoV-2 spike protein and loaded with small interfering RNA (siRNA) green fluorescent protein (GFP) could specifically target hACE2 receptors on GFP-expressing A549 lung adenocarcinoma cells and dramatically diminish the level of GFP expressed in the cells.