Self-assembling CpG DNA nanoparticles for efficient antigen delivery and immunostimulation

DNA containing unmethylated deoxycytidylyl-deoxyguanosine (CpG) dinucleotides (CpG DNA) is a potent stimulator of immune responses through triggering of Toll-like receptor 9 (TLR9). In the present study, we synthesized cholesterol-modified CpG oligodeoxynucleotide (Chol-CpG ODN) and investigated its ability to form nanoparticles by self-assembling, then examined their immunostimulatory activity and potency to deliver antigens to antigen presenting cells (APCs). Chol-CpG ODN spontaneously formed particles in aqueous solutions. Cholesterol modification increased the stability of ODN in serum. Chol-CpG ODN was efficiently taken up by mouse macrophage-like RAW264.7 cells and induced a large amount of tumor necrosis factor-α compared with unmodified CpG ODN. Then, ovalbumin (OVA), a model antigen, was incorporated into Chol-CpG ODN nanoparticles. Cholesterol-modified GpC ODN (Chol-GpC ODN) was used to assess the importance of CpG motif on the antigen-specific immune response. Vaccination of mice with OVA/Chol-CpG ODN induced high level interferon-γ production from splenocytes. Furthermore, a high serum level of OVA-specific immunoglobulin G2a was observed in mice receiving OVA/Chol-CpG ODN. Neither CpG ODN nor Chol-GpC ODN was effective at all. These results indicate that self-assembling nanoparticles of Chol-CpG ODN are effective for inducing antigen-specific immune responses because of the high immunostimulatory activity, ability to incorporate antigens and tropism to APCs.