Salivary Exosomes Confer Exogenous mRNA Delivery in Oral Epithelial Cells

Objectives: Calprotectin (S100A8/A9) is an antimicrobial protein produced in the gingiva. In a murine model for periodontitis, S100A8/A9 modifies the plaque microbiota, attenuates inflammation, and reduces alveolar bone loss. We hypothesize that delivery of calprotectin mRNA in vivo will slow the progression of periodontitis. Efficient mRNA delivery to the oral epithelium, however, remains challenging. To overcome this challenge, we compare polymeric delivery vehicles to exosomes derived from a variety of sources for efficiency of mRNA delivery into oral epithelial cells in vitro.
Methods: Physical properties of exosomes and exosome-mRNA complexes were assessed using dynamic light scattering and zeta potential measurements (Malvern Zetasizer). Exosomes collected by ultracentrifugation. Transfections: 50k cells/well, 1.5 µg mRNA/well, 10 ug exosomes/well. Gene expression was assessed using FACs (eGFP reporter) or Promega Luciferase Assay System (luciferase reporter). Protein content was assessed using western blotting and Licor Revert total protein stain.
Results: Salivary exosomes (SE) have a Z_ave size of 105 nm while exosomes derived from oral epithelial cells are 83 nm. SE are more disperse and show different protein profiles than exosomes derived from cultured oral epithelial cells. Mixing with mRNA leads to partial aggregation of exosomes. mRNA loaded exosomes can be recovered by centrifugation and resuspend readily in PBS. After resuspension they exhibit a Z_ave size of 115 nm. When complexed with mRNA, SE efficiently transfect oral epithelial cells (Tr146) in vitro (60% positive cells). Transfection with exosomes is essentially non-toxic (95% cell survival).
Conclusions: SE are heterogeneous in size, indicating a more complex mixture of particles than exosomes derived from cell culture. Their ability to transfect epithelial cells suggests that SE are promising vehicles for gene delivery to the oral epithelium. The data suggest that exosome-mediated delivery of S100A8/A9 mRNA cargo could augment production of S100A8/A9 in oral epithelial cells in vitro and in vivo.