Putting some jam on the sandwich: the addition of cationic peptide-encapsulated mRNA turns the layer-by-layer technology into a versatile mRNA vaccine platform.

In recent years, mRNA-based therapeutics have proven to be powerful tools in combatting infectious diseases. This became particularly evident during the COVID-19 pandemic, where mRNA-based vaccines showcased their true potential. Current research mainly focuses on lipid nanoparticles, leaving many alternative delivery systems unexplored. Developing a successful mRNA formulation requires selecting the most suitable system, which depends on the payload size, target cells and the intended outcome. While lipid nanoparticles have demonstrated safety and immunogenicity, their potential for functionalization is complex and limited. Therefore, there is a need to design more customizable formulations.
The layer-by-layer technique (LbL), introduced by Decher et al. in 1998, is a well-known method that involves the iterative disposition of positively and negatively charged polyelectrolyte layers around a CaCO3 core to create submicroparticles (SubP). The main advantages of this platform include its low cost, easy manufacturing process and broad application potential. However, despite efficient uptake, we observed no protein translation when mRNA was conjugated with these particles, either in the core or in between the layers. Our group previously demonstrated the efficiency of the histidine-rich cationic peptide LAH4-L1, in formulating mRNA resulting in efficient in vitro and in vivo translation and the subsequent induction of ovalbumin-specific CD8+ T cell responses. We, therefore, hypothesized that combining LAH4-L1 with SubP referred to as SubP-L1, would enhance the translation efficiency of the LbL SubP.
Using both conventional and self-amplifying mRNA, SubP-L1 achieves comparable translation efficiency and antigen presentation with LAH4-L1 in both murine dendritic cell line (DC2.4 cells) and primary cells (murine bone marrow- and human monocyte-derived dendritic cells). Moreover, in vivo immunization with SubP-L1 resulted in enhanced proliferation of ovalbumin-specific CD8+ T cells in lymphoid tissue compared to LAH4-L1. Lastly, these particles have demonstrated that they maintain their physicochemical properties and protein translation for at least 7 days at both room temperature and at 4°C, showcasing their stability, and thus highlighting their potential as a robust mRNA vaccine platform.
Overall, these promising results suggest that the modified layer-by-layer platform presented here holds the potential to significantly advance the development of highly effective mRNA-based vaccines.