Fabrication of RIG-I-Activating Nanoparticles for Intratumoral Immunotherapy via Flash Nanoprecipitation.

Intratumoral immunotherapy is a promising strategy for stimulating local and systemic antitumor immunity while eliminating or reducing immune-related adverse events often attendant to systemic administration. Activation of the cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I) at tumor sites stimulates innate immunity that can potentiate a T cell-dependent adaptive antitumor immune response. However, the activity and efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by poor in vivo stability, rapid degradation, limited cellular uptake, and inefficient cytosolic delivery. To overcome these challenges, we developed RIG-I-activating nanoparticles (RANs) assembled using a flash nanoprecipitation (FNP) process to load a potent stem-loop 3pRNA (SLR) RIG-I agonist into endosome-destabilizing polymeric nanoparticles. We leveraged FNP to induce turbulent micromixing among a corona-forming poly(ethylene glycol)--(dimethylaminoethyl methacrylate--butyl methacrylate) (PEG-DB) diblock copolymer, a hydrophobic core-forming DB counterpart, and an SLR RIG-I agonist, resulting in the self-assembly of densely loaded nanoparticles that promoted endosomal escape and cytosolic delivery of 3pRNA cargo. Through optimization of polymer properties and inlet feed ratios, we developed RANs with high and improved loading efficiency and increased serum stability relative to a previously reported micelleplex formulation assembled via electrostatic complexation with PEG-DB polymers. We found that optimized RANs exhibited potent immunostimulatory activity in vitro and in vivo when delivered intratumorally. As a result, in preclinical models of MC38 colon cancer and B16.F10 melanoma, intratumoral administration of RANs suppressed tumor growth and increased survival time relative to vehicle controls. Collectively, this work demonstrates that FNP can be harnessed as a versatile and scalable process for the efficient loading of nucleic acids into polymeric nanoparticles and highlights the potential of RANs as a translationally promising platform for intralesional cancer immunotherapy.