3D RNA nanocage for encapsulation and shielding of hydrophobic biomolecules to improve the in vivo biodistribution

Ribonucleic acid (RNA) nanotechnology platforms have the potential of harboring therapeutics for in vivo delivery in disease treatment. However, the nonspecific interaction between the harbored hydrophobic drugs and cells or other components before reaching the diseased site has been an obstacle in drug delivery. Here we report an encapsulation strategy to prevent such nonspecific hydrophobic interactions in vitro and in vivo based on a self-assembled three-dimensional (3D) RNA nanocage. By placing an RNA three-way junction (3WJ) in the cavity of the nanocage, the conjugated hydrophobic molecules were specifically positioned within the nanocage, preventing their exposure to the biological environment. The assembly of the nanocages was characterized by native polyacrylamide gel electrophoresis (PAGE), atomic force microscopy (AFM), and cryogenic electron microscopy (cryo-EM) imaging. The stealth effect of the nanocage for hydrophobic molecules in vitro was evaluated by gel electrophoresis, flow cytometry, and confocal microscopy. The in vivo sheathing effect of the nanocage for hydrophobic molecules was assessed by biodistribution profiling in mice. The RNA nanocages with hydrophobic biomolecules underwent faster clearance in liver and spleen in comparison to their counterparts. Therefore, this encapsulation strategy holds promise for in vivo delivery of hydrophobic drugs for disease treatment.