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.

Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion.

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.

Inhibition of surgical trauma-enhanced peritoneal dissemination of tumor cells by human catalase derivatives in mice.

Surgical trauma, which is inevitably associated with the surgical removal of cancer, has been reported to accelerate tumor metastasis. The close association of reactive oxygen species with the trauma and tumor metastasis supports the possibility of using antioxidants for the inhibition of metastasis. To inhibit surgical trauma-enhanced peritoneal dissemination, human catalase (hCAT) derivatives, i.e., hCAT-nona-arginine peptide (hCAT-R9) and hCAT-albumin-binding peptide (hCAT-ABP), were designed to increase the retention time of the antioxidant enzyme in the abdominal cavity after intraperitoneal administration. Both (125)I-labeled derivatives showed significantly prolonged retention in the cavity compared to (125)I-hCAT. Cauterization of the cecum of mice with a hot iron, an experimental model of surgical trauma, induced abdominal adhesions. In addition, cauterization followed by colon26 tumor cell inoculation increased lipid peroxidation in the cecum and mRNA expression of molecules associated with tissue repair/adhesion and inflammation in the peritoneum. hCAT derivatives significantly suppressed the increased mRNA expression. The cauterization also increased the number of tumor cells in the abdominal organs, and the number was significantly reduced by hCAT-R9 or hCAT-ABP. These results indicate that hCAT-R9 and hCAT-ABP, both of which have a long retention time in the peritoneal cavity, can be effective at inhibiting surgery-induced peritoneal metastasis.

Enhanced generation of cytotoxic T lymphocytes by heat shock protein 70 fusion proteins harboring both CD8(+) T cell and CD4(+) T cell epitopes.

Heat shock protein 70 (Hsp70) can be a potent carrier of antigens because it is effectively delivered to antigen presenting cells (APCs) and activates innate immunity. To induce a potent cytotoxic T lymphocyte (CTL) response, a Hsp70 fusion protein harboring both CD8(+) and CD4(+) T cell epitopes was developed based on the recent understanding of the importance of the role of CD4(+) T cells in inducing the CTL response following vaccination. OVA(257-264) (pepI) and OVA(323-339) (pepII) were selected as the CD8(+) and CD4(+) T cell epitope of a model antigen, ovalbumin (OVA), respectively. Hsp70 and its fusion proteins, Hsp70-pepI, pepII-Hsp70, Hsp70-pepII and pepII-Hsp70-pepI, were developed. pepII-Hsp70 and pepII-Hsp70-pepI were effectively presented on MHC class II of macrophages compared with Hsp70-pepII, suggesting that pepII conjugation to the N-terminus of Hsp70 is better than the C-terminus for more effective MHC class II antigen presentation. Immunization with pepII-Hsp70-pepI resulted in a higher CTL activity than immunization with the mixture of Hsp70-pepI and pepII-Hsp70. Furthermore, pepII-Hsp70-pepI exhibited a greater antitumor effect in the mice bearing EG7 tumor cells than the physical mixture of Hsp70-pepI and pepII-Hsp70. In addition, immunization with the DCs pulsed with the fusion proteins also suggested that APCs which present both pepI and pepII can induce the highest CTL generation. These results demonstrated that Hsp70 fusion protein harboring both pepI and pepII is a useful option for Hsp70-based antigen delivery systems.

Injection site-dependent induction of immune response by DNA vaccine: comparison of skin and spleen as a target for vaccination.

BACKGROUND: The antigen-specific immune response is dependent not only on the properties of the antigens, but also on their encounter with antigen-presenting cells. A previous study showed that the spleen produced a large amount of transgenes after direct tissue injection of plasmid DNA. In addition, the spleen is the largest organ in the lymphatic system and contains a variety of types of immune cells, including lymphocytes, macrophages and dendritic cells. Thus, it can be a promising target for DNA vaccination. METHODS: Tissue-dependent properties of transgene expression were examined using a plasmid vector expressing firefly luciferase. Mice received injections of pCMV-Luc into the dorsal skin or spleen followed by electroporation, and the luciferase activity was measured 6 h after injection. Then, plasmids expressing a model antigen ovalbumin (pCMV-OVA) or its typical major histocompatibility complex class I-restricted epitope SIINFEKL (pPep-ER) were injected into C57BL/6 mice twice at an interval of 1 week. Seven days after the second immunization, OVA-specific humoral and cellular immune responses were evaluated. RESULTS: The spleen produced a larger amount of transgenes than the skin after direct tissue injection of plasmid DNA. However, intradermal injection of plasmid DNA resulted in a larger amount of OVA-specific antibodies and a greater cytotoxic T lymphocyte response compared to intrasplenic injection. In addition, intradermal immunization with either pCMV-OVA or pPep-ER generated more protective effects against EG7-OVA tumor challenge. CONCLUSIONS: The results obtained in the present study indicate that the spleen is unlikely to be a good target for immunization despite the presence of a large number of lymphocytes and efficient production of transgenes.

Control of hypoxia-induced tumor cell adhesion by cytophilic human catalase.

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.