DNA nanotechnology-based nanosystems and macrosystems have attracted much attention in the biomedical research field. The nature of DNA endows these systems with biodegradable, biocompatible, and immunomodulatory properties. Here, we present an injectable hydrogel system that consists only of chemically synthesized short DNA strands, water, and salts. Several preparations of polypod-like structured DNA, or polypodna, were designed, including tri-, tetra-, penta- and hexapodna, as the building blocks of self-gelling DNA hydrogel. Under physiological conditions, properly designed polypodna preparations formed a hydrogel. The analysis of the modulus data of the hydrogel consisting of two sets of hexapodna preparations showed that this injectable hydrogel was reorganized at a time scale of 0.25s. Then, DNA hydrogel containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides was used to stimulate innate immunity through Toll-like receptor 9, the receptor for CpG DNA. Gel formation significantly increased the activity of immunostimulatory CpG DNA, retarded the clearance after intradermal injection into mice, and increased the immune responses to ovalbumin (OVA) incorporated into the hydrogel as a model antigen. OVA/CpG DNA hydrogel induced much less local or systemic adverse reactions than OVA injected with complete Freund's adjuvant or alum. GpC DNA hydrogel containing no CpG sequences was less effective, indicating the importance of immunomodulation by CpG DNA hydrogel. Thus, we have created an efficient system for sustained delivery of antigens or other bioactive compounds.

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.

Previous studies indicate that immunostimulatory DNA‐based injectable hydrogels harboring unmethylated cytosine‐phosphate‐guanine (CpG) dinucleotides meet the requirements of an effective antigen delivery system, including safety, biodegradability, ease of administration, and stimulation of the innate immune system. However, rapid release of the model antigen ovalbumin (OVA) from the hydrogel limits its potential. Here, the aim is to achieve sustained OVA release from a DNA hydrogel through cationization of the antigen. Ethylenediamine (ED)‐conjugated cationized OVA (ED‐OVA), but not OVA, forms a complex with hexapod‐like structured DNA, a component of the DNA hydrogel. The release of ED‐OVA from the hydrogel is significantly slower than that of OVA. ED‐OVA mixed with CpG DNA hydrogel efficiently binds to mouse dendritic DC2.4 cells and results in high antigen presentation. Intratumoral injections of ED‐OVA/CpG DNA hydrogel significantly delays tumor growth of OVA‐expressing EG7‐OVA cells in mice. Then, a cationic OVA peptide antigen (R8‐L2‐pepI) consisting of an OVA MHC class I epitope, octaarginine, and a linker is designed. Intratumoral injections of R8‐L2‐pepI/CpG DNA hydrogel eradicate tumors in five out of six mice. Thus, it is concluded that a vaccine consisting of immunostimulatory CpG DNA hydrogel and cationized antigens can be effective for cancer immunotherapy.