Immunostimulation of Fibrous Nucleic Acid Nanoparticles Can be Modulated through Aptamer-Based Functional Moieties: Unveiling the Structure-Activity Relationship and Mechanistic Insights.

Fibrous nanomaterials containing silica, titanium oxide, and carbon nanotubes are notoriously known for their undesirable inflammatory responses and associated toxicities that have been extensively studied in the environmental and occupational toxicology fields. Biopersistance and inflammation of "hard" nanofibers prevent their broader biomedical applications. To utilize the structural benefits of fibrous nanomaterials for functionalization with moieties of therapeutic significance while preventing undesirable immune responses, researchers employ natural biopolymers─RNA and DNA─to design "soft" and biodegradable nanomaterials with controlled immunorecognition. Nucleic acid nanofibers have been shown to be safe and efficacious in applications that do not require their delivery into the cells such as the regulation of blood coagulation. Previous studies demonstrated that unlike traditional therapeutic nucleic acids (e.g., CpG DNA oligonucleotides) nucleic acid nanoparticles (NANPs), when used without a carrier, are not internalized by the immune cells and, as such, do not induce undesirable cytokine responses. In contrast, intracellular delivery of NANPs results in cytokine responses that are dependent on the physicochemical properties of these nanomaterials. However, the structure-activity relationship of innate immune responses to intracellularly delivered fibrous NANPs is poorly understood. Herein, we employ the intracellular delivery of model RNA/DNA nanofibers functionalized with G-quadruplex-based DNA aptamers to investigate how their structural properties influence cytokine responses. We demonstrate that nanofibers' scaffolds delivered to the immune cells using lipofectamine induce interferon response via the cGAS-STING signaling pathway activation and that DNA aptamers incorporation shields the fibers from recognition by cGAS and results in a lower interferon response. This structure-activity relationship study expands the current knowledge base to inform future practical applications of intracellularly delivered NANPs as vaccine adjuvants and immunotherapies.