Engineering immunostimulatory nanocarriers: TLR7-agonist conjugated poly(beta-amino ester) mRNA delivery systems.

Messenger RNA (mRNA) technology serves as a powerful foundation for novel vaccines and treatments. Ensuring the effective delivery of RNA medicines requires vectors due to the poor cellular association of naked mRNA and its susceptibility to degradation by endogenous nucleases. Recently, nanoparticle-based carriers have attracted significant attention, particularly poly(beta-amino esters) (PBAEs), which have shown promise as mRNA delivery systems. The first generation of nanoparticle-encapsulated mRNA vaccines has been highly successful in protecting against severe COVID-19 disease, but the durability of the immune response remains short; hence, there is a need for delivery systems that enhance the robustness of the immune response. In this study, we report the development of a TLR7-adjuvanting PBAE through the direct conjugation of Loxoribine, a potent TLR7 agonist. We observed that the Loxoribine-conjugated PBAE was able to condense self-amplifying RNA (saRNA) efficiently into small (100-200 nm) polyelectrolyte nanoparticles with complete incorporation of the nucleic acid. Formulations specifically at a w/w ratio of 128 showed high transfection in HEK293T and DC2.4 cell lines with good cytotoxicity profiles. In addition, TLR activation assays using TLR7 reporter HEK-Blue cells showed that Loxoribine-conjugated formulations can efficiently agonise TLR7, with some synergy observed between Loxoribine and the delivered saRNA. Although potent activation was observed in model cell lines, evaluation in primary bone marrow-derived dendritic cells showed minimal upregulation of CD86, an established activation marker, and a transfection efficiency of less than 2%. Consequently, these findings suggest the potential of adjuvanted PBAE nanoparticles for enhanced RNA vaccines; however, further development is needed to improve their activity in primary immune cells.