Layer-by-Layer Assembly of Polymeric Nanoparticles with Heparin-RBD Complexes as an Adjuvant for SARS-CoV-2 Protein-Based Vaccines

Multilayered polymeric particles work as carriers of compounds with controlled release triggered by the layer's disassembly, allowing chemical stability along with transport through the bloodstream and tissues. Here, we present versatile multilayered polymeric particles as a vaccine approach against COVID-19 based on coupling a recombinant form of the receptor binding domain (RBD) antigen of the SARS-CoV-2 Spike proteins. The particle core comprises poly(D,L-lactide) coated with Triton X-100 and polyethylenimine, prepared by nanoemulsion/solvent evaporation method and functionalized with heparin by layer-by-layer (PLTP-H). The recombinant RBD is anchored to the nanoparticles by the negative outer layer (PLTP-H-RBD). The nanoparticles reached a complexation efficiency of 95% for the RBD and a final hydrodynamic diameter of 140 nm with a zeta potential of -46 mV. No toxicity was recorded with the nanoparticles using cultured mammalian cells or after inoculation into mice. The immunization of mice with antigen-loaded nanoparticles induced strong adjuvant effects on the serum levels of RBD-specific IgG antibodies, which were capable of neutralizing the virus under in vitro conditions. Furthermore, PLTP-H-RBD conferred enhanced protective immunity to mice challenged with the SARS-CoV-2 virus. These findings represent a proof of concept for the use of PLTP-H as a delivery and adjuvant strategy for recombinant vaccine antigens aiming at the induction of neutralizing antibodies.