Lipid nanoparticle-formulated DNA acts as a potent immune modulator for cancer immunotherapy through interferon signaling pathways

<b>Rationale:</b> Plasmid DNA (pDNA) delivered by lipid nanoparticles (LNPs) represents a promising strategy for cancer immunotherapy, offering both stability of nucleic acids and efficient intracellular delivery. This study aimed to evaluate the stability and immunotherapeutic potential of LNP/pDNA formulations and to define the mechanisms underlying their antitumor activity. <b>Methods:</b> LNP/pDNA complexes were prepared by a microfluidic mixer system. Encapsulation efficiency, particle size, and transfection capacity were determined at different time points following formulation to assess physicochemical stability. <i>In vivo</i> antitumor efficacy was evaluated using intratumoral and intramuscular administration in murine tumor models. Mechanistic studies included cytokine profiling, transcriptomic analysis of tumors, and immune cell depletion experiments. Mouse models deficient in TLR9 and interferon signaling pathways were employed to dissect signaling pathway contributions. <b>Results:</b> LNP/pDNA formulations retained encapsulation efficiency and size uniformity after prolonged storage and maintained effective gene delivery. Both intratumoral and intramuscular administration suppressed tumor growth, with local delivery showing superior efficacy. LNP/pDNA activated cytosolic DNA-sensing pathways and induced robust proinflammatory cytokine production. Transcriptomic analysis revealed strong type I and II interferon responses and upregulation of immune effector pathways. Depletion studies confirmed that antitumor effects were dependent on CD8⁺ T cells and NK cells but independent of neutrophils and monocytes. Notably, therapeutic efficacy was preserved in TLR9-deficient mice but lost in mice lacking both type I and II interferon signaling. <b>Conclusions:</b> LNP/pDNA induces potent antitumor immunity through activation of IFN-dependent, TLR9-independent pathways, engaging both innate and adaptive immune responses. These findings support LNP/pDNA as a stable, effective platform for cancer immunotherapy.