In Vitro and In Vivo Stimulation of Toll-Like Receptor 9 by CpG Oligodeoxynucleotides Incorporated Into Polypod-Like DNA Nanostructures

Cytosine-phosphate-guanine (CpG) DNA is known to increase the potency of vaccines. Here, in vitro and in vivo stimulation of toll-like receptor 9 by CpG DNA incorporated into polypod-like DNA nanostructures was evaluated by measuring the levels of tumor necrosis factor alpha released from macrophage-like RAW 264.7 cells and plasma interleukin (IL)-12p40 in vivo following intravenous injection into mice. Phosphodiester CpG1668 was selected as the CpG DNA, and tripodna and hexapodna, which were CpG1668-containing tripod and hexapod-like DNA nanostructures, respectively, were designed. CpG-tripodna and CpG-hexapodna induced tumor necrosis factor alpha release from RAW 264.7 cells about 10- and ∼30-fold higher than single-stranded CpG1668 (CpG-SS). Moreover, in all cases examined, plasma IL-12p40 concentrations increased after intravenous injection into mice, with peak levels depending on the samples and the doses. The area under the plasma concentration-time curves indicated that the CpG-hexapodna was approximately 20-fold more efficient in inducing IL-12p40 production than CpG-SS. The efficiency of CpG-tripodna and CpG-hexapodna to increase the potency of CpG-SS in vivo was comparable to that observed in cultured RAW 264.7 cells. These results provide experimental evidence that in vitro studies can be used to estimate the in vivo immunostimulatory activity of CpG DNA incorporated into DNA nanostructures.