Dynamic DNA-Based Nanoadjuvants for TLR9 Clustering and Innate Immune Activation in Dendritic Cells

The regulation of toll-like receptor (TLR) clustering is a pivotal strategy for enhancing innate immune responses, but the development of methods to precisely control receptor assembly remains challenging. Herein, we present a dynamic, DNA-based nanoadjuvant that triggers TLR9 clustering for potent innate immune activation in dendritic cells (DCs) via in situ assembly in lysosomes. This nanoadjuvant integrates CpG oligonucleotides (TLR9 ligands) and cytosine-rich DNA sequences (acid-responsive sequences) into a polymeric nanoframework via a cascade hybridization chain reaction. Upon lysosomal internalization, the nanoadjuvants form large-sized aggregates through cytosine protonation-induced i-motif formation, a process driven by the acidic lysosomal environment. This assembly consumes lysosomal protons, thus reducing lysosomal acidity and attenuating hydrolase activity, leading to enhanced intralysosomal retention of nanoadjuvants. Moreover, the nanoadjuvant aggregates promote CpG ODN contact with the lysosomal membrane, thereby facilitating prolonged ligand-receptor interactions and efficient TLR9 clustering. Consequently, the nanoadjuvant aggregates induce efficient DC maturation, secretion of cytokines, and T-cell proliferation, culminating in robust antitumor immunity both in vitro and in vivo. Our approach offers a novel strategy to manipulate receptor assembly using an environmentally stimulus-responsive system, holding significant promise for vaccine adjuvants and cancer immunotherapy.