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.

The application of DNA as a functional material such as DNA hydrogel has attracted much attention. Despite an increasing interest, the high cost of DNA synthesis is a limiting factor for its utilization. To reduce the cost, we report here a highly efficient amplification technique for polypod-like structured DNA (polypodna) with adhesive ends that spontaneously forms DNA hydrogel. Two types of polypodna with three (tripodna) and four (tetrapodna) pods were selected, and a template oligodeoxynucleotide, containing a tandem sequence of a looped tripodna or tetrapodna, respectively, along with restriction enzyme (TspRI) sites, was designed. The template was circularized using T4 DNA ligase, and amplified by rolling circle amplification (RCA). The RCA product was highly viscous and resistant to restriction digestion. Observation under an electron microscope revealed microflower-like structures. These structures were composed of long DNA and magnesium pyrophosphate, and their treatment with EDTA followed by restriction digestion with TspRI resulted in numerous copies of polypodna with adhesive ends, which formed a DNA hydrogel. Thus, we believe this technique provides a new approach to produce DNA nanostructures, and helps in expanding their practical applications.

Hypoxia-induced reactive oxygen species (ROS)-mediated expression of a variety of genes in endothelial cells has been suggested to be involved in abnormal cell adhesion. To prevent this by accelerated binding of catalase to endothelial cells, human catalase (hCAT), an enzyme catalyzing the decomposition of hydrogen peroxide, was fused with three repeats of arginine-glycine-aspartic acid peptide or nona arginine peptide at the C-terminal to obtain hCAT-(RGD)3 and hCAT-R9, respectively. Human CAT and its derivatives were expressed in yeast Pichia pastoris and purified. The specific activity and secondary structure of hCAT-(RGD)3 and hCAT-R9 were close to those of hCAT, but these derivatives showed higher binding to the mouse aortic vascular endothelial cell line MAEC than hCAT, indicating that they are cytophilic derivatives. Hypoxic treatment of MAEC increased the intracellular ROS level, the binding of mouse melanoma cells, and the activity of transcription factors, hypoxia inducible factor-1 and nuclear factor-kappaB. hCAT-(RGD)3 or hCAT-R9 efficiently inhibited these changes compared with hCAT. These results indicate that cytophilic hCAT-(RGD)3 and hCAT-R9 are effective in inhibiting hypoxia-induced tumor cell adhesion to endothelial cells.