Encapsulation of Recombinant ACE-2 in Chitosan Nanoparticles for Treatment of SARS-CoV-2 Infection

COVID-19 infection continues globally with frequent emergence of unfamiliar SARS-CoV-2 variants acting to impair immunity conferred by vaccines. The competitive binding of SARS-CoV-2 spike proteins by angiotensin-converting enzyme 2 (ACE-2) to mimetic and act as a de-coy over that by native ACE-2 receptors on healthy human cells re-mains a practical approach to lessen viral spread. In this study, a therapeutic strategy was developed that targeted gastrointestinal SARS-CoV-2 infection using ACE-2 encapsulated in chi-tosan/tripolyphosphate cross-linked nanoparticles (NPs). Optimization conditions were determined by varying pH (4.0-6.5) and chitosan: ACE-2 mixing ratios (1:1, 1.5:1, 2:1, 2.5:1, 3:1), followed by choice of spray-drying (SD), freeze-drying (FD), or spray-freeze drying (SFD) with varying mannitol concentrations (0, 1:1, and 5:1 of total weight). The optimal formulation was achieved using a pH 5.5 with a mixing chitosan-ACE-2 ratio of 2:1; where ACE-2 loaded NPs had an average particle size of 303.7 nm, polydispersity index (PDI) of 0.21, encapsula-tion efficiency (EE) of 98.4%, zeta potential of 6.8 mV, and ACE-2 loading content (LC) of 28.4%. In general, all drying methods main-tained the spherical shape of the NPs with varying mannitol concen-tration having a significant (P<0.05) effect. After reconstitution, all SD samples had a relatively low yield rate, but the ACE-2 NPs dehydrated specifically by SFD required a lower amount of added mannitol (1:1 of its total weight) and produced a higher yield rate (P<0.05) and similar PDI and EE values, along with relatively good particle size and LC. This formulation also produced a high ACE-2 release and uptake in differentiated Caco-2 cells; thus, representing an effective ACE-2 en-capsulation procedure for use with dry powders.