DNA nanotechnology-based composite-type gold nanoparticle-immunostimulatory DNA hydrogel for tumor photothermal immunotherapy.

Success of tumor photothermal immunotherapy requires a system that induces heat stress in cancer cells and enhances strong anti-tumor immune responses. Here, we designed a composite-type immunostimulatory DNA hydrogel consisting of a hexapod-like structured DNA (hexapodna) with CpG sequences and gold nanoparticles. Mixing of the properly designed hexapodna and oligodeoxynucleotide-modified gold nanoparticles resulted in the formation of composite-type gold nanoparticle-DNA hydrogels. Laser irradiation of the hydrogel resulted in the release of hexapodna, which efficiently stimulated immune cells to release proinflammatory cytokines. Then, EG7-OVA tumor-bearing mice received an intratumoral injection of a gold nanoparticle-DNA hydrogel, followed by laser irradiation at 780 nm. This treatment increased the local temperature and the mRNA expression of heat shock protein 70 in the tumor tissue, increased tumor-associated antigen-specific IgG levels in the serum, and induced tumor-associated antigen-specific interferon-γ production from splenocytes. Moreover, the treatment significantly retarded the tumor growth and extended the survival of the tumor-bearing mice.

Polypod-Shaped DNAs: Small-Angle X-ray Scattering and Immunostimulatory Activity.

We explored in detail the relationship between the structure in aqueous solution and immunostimulatory activity of polypod-shaped DNAs, called polypodnas. The polypodnas were constructed using 3-6 oligodeoxynucleotides (ODNs) to obtain tri-, tetra-, penta-, and hexapodna, each of which had 3, 4, 5, and 6 arms made of double-stranded DNA, respectively. A highly potent immunostimulatory CpG sequence was included into each of the polypodnas. Synchrotron X-ray scattering analysis showed that the double-stranded DNA arms of all of the polypodnas adopted a B-form DNA conformation. The analysis also suggested that some nucleotides in the central parts of pentapodna and hexapodna did not form base pairs, whereas those of tripodna and tetrapodna all formed base pairs. This difference would occur because of an increase in steric hindrance and electrical repulsion with increasing number of arms. The pentapodna and hexapodna induced a large amount of tumor necrosis factor α-release from macrophage-like cells compared with the tripodna and tetrapodna, suggesting that the partly loosened DNA in polypodna with many arms is advantageous for exposing the immunostimulatory sequences of the polypodna.

X-ray scattering from immunostimulatory tetrapod-shaped DNA in aqueous solution to explore its biological activity-conformation relationship.

We carried out synchrotron X-ray scattering experiments from four DNA supermolecules designed to form tetrapod shapes; these supermolecules had different sequences but identical numbers of total base pairs, and each contained an immunostimulatory CpG motif. We confirmed that the supermolecules did indeed form the expected tetrapod shape. The sample that had the largest radius of gyration (Rg) induced the most cytokine secretion from cultured immune cells. Structural analysis in combination with a rigid tetrapod model and an atomic scale DNA model revealed that the larger Rg can be ascribed to dissociation of the DNA double strands in the central connecting portion of the DNA tetrapod. This finding suggests that the biological activity is related to the ease with which single DNA strands can be formed.

Increased immunostimulatory activity of polypod-like structured DNA by ligation of the terminal loop structures.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-guanine (CpG) dinucleotides can be increased by converting it into branched structures. These structures could be stabilized by ligating the 5'- and 3'-ends to form a closed loop with no terminal ends. To further increase the ability of branched DNA assemblies to induce cytokines, a series of tetrapod-like structured DNA, or tetrapodna, were designed using four 48-base oligodeoxynucleotides (ODNs). All these preparations were designed to have the same sequence except for the nick sites, and all the ODNs of one of the tetrapodna preparations were ligated to obtain circular tetrapodna. The nick site significantly influenced the formation of the structure and melting temperature (Tm), but hardly affected the enzymatic stability of the tetrapodna preparations. Circular tetrapodna exhibited a significantly higher Tm and was more stable in mouse serum than its non-ligated counterparts. The amounts of cytokines released from macrophage-like RAW264.7 cells or dendritic DC2.4 cells after addition of circular tetrapodna were not significantly higher than those after addition of other tetrapodna preparations under conditions when no serum was present. However, when serum was present, circular tetrapodna induced the greatest amount of tumor necrosis factor-α, indicating that circular tetrapodna is effective in inducing cytokines under conditions where DNA-degrading enzymes are present. The cellular association of tetrapodna preparations was almost unaffected by ligation of the terminal ends. These results indicate that circular tetrapodna with no terminal ends is more effective than its non-ligated counterparts in the presence of serum.

Design and development of nanosized DNA assemblies in polypod-like structures as efficient vehicles for immunostimulatory CpG motifs to immune cells.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG motifs, was significantly increased by the formation of Y-, X-, or dendrimer-like multibranched shape. These results suggest the possibility that the activity of CpG DNA is a function of the structural properties of branched DNA assemblies. To elucidate the relationship between them, we have designed and developed nanosized DNA assemblies in polypod-like structures (polypod-like structured DNA, or polypodna for short) using oligodeoxynucleotides (ODNs) containing CpG motifs and investigated their structural and immunological properties. Those assemblies consisting of three (tripodna) to eight (octapodna) ODNs were successfully obtained, but one consisting of 12 ODNs was not when 36-mer ODNs were annealed under physiological sodium chloride concentration. High-speed atomic force microscopy revealed that these assemblies were in polypod-like structures. The apparent size of the products was about 10 nm in diameter, and there was an increasing trend with an increase in ODN length or with the pod number. Circular dichroism spectral data showed that DNA in polypodna preparations were in the B-form. The melting temperature of polypodna decreased with increasing pod number. Each polypodna induced the secretion of tumor necrosis factor-α and interleukin-6 from macrophage-like RAW264.7 cells, with the greatest induction by those with hexa- and octapodna. Increasing the pod number increased the uptake by RAW264.7 cells but reduced the stability in serum. These results indicate that CpG DNA-containing polypodna preparations with six or more pods are a promising nanosized device with biodegradability and high immunostimulatory activity.