Development and evaluation of nanoparticular vaccine formulations against COVID-19

The 2019 coronavirus disease outbreak (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has demonstrated the need for rapid, effective and safe vaccines worldwide. In this study, subunit protein nanovaccine was developed and it is aimed to improve the problems encountered in the development and application of vaccines by examining the effects of formulation-related parameters (preparation method, material used and method steps) on the prepared nanoparticle carrier system. In this study, nanoparticular subunit protein vaccine has been studied and the SARS-CoV-2 spike protein receptor binding domain (RBD) has been used as a protein. The RBD protein was chosen because it interacts directly with the ACE2 receptor on host cells and contains highly neutralizing epitopes. Modified water/oil/water (W1/O/W2) emulsion-solvent evaporation technique has been used for the preparation of RBD-loaded PEG-PCL nanoparticles; the nanoparticles has been evaluated in terms of particle size and distribution, zeta potential and encapsulation efficiency. The morphology of the nanoparticles was visualized by transmission electron microscopy (TEM). Then, in vitro release studies has been carried out with optimized nanoparticles. SDS-PAGE study has been performed to evaluate the effect of the manufacturing process on the structural integrity of the antigen and in vitro release samples. The physical stability of nanoparticles has been determined by performing physical stability studies for 1 month. MTT analysis performed using the L929 cell line determined % cell viability, and Nitric Oxide determination was performed using the J774 mouse macrophage cell line to observe the in vitro activity of RBD-loaded nanoparticles. In this research, the particle size, distribution and zeta potential of RBD-NPs were found to be in the targeted range (particle size <300 nm, PDI <0.3, Zeta potential ±10–15 mV) and the antigen was effectively encapsulated. TEM images revealed that the nanoparticles were spherical. NP formulations released 50% of the RBD within 4 hours, and the release continued for up to 96 hours, reaching a plateau when approximately 68% of the active substance was released. SDS-PAGE results showed that the production process did not cause any changes in the structure of the antigen and that the antigen remained stable throughout the release period. As a result of stability studies, it was found that NPs remained physically stable at +4ºC and -20°C with the addition of trihalose. As a result of cell viability and in vitro biological activity studies, it was determined that it was non-toxic and showed immunostimulatory activity even at the highest concentrations after 72 hours of incubation. Within the scope of these findings, subunit protein nanovaccine has been successfully formulated and characterized. It has been seen that the subunit protein nanovaccine system, which was developed as an alternative to traditional vaccines, has great potential in future viral outbreaks such as COVID-19.