Lasso peptide microcin J25 variant containing RGD motif as a PET probe for integrin a v ß 3 in tumor imaging

Microcin J25 (MccJ25), a lasso peptide, has a unique 3-D interlocked structure that provides high stability under acidic conditions, at high temperatures, and in the presence of proteases. In this study, we generated a positron emission tomography (PET) probe based on MccJ25 analog with an RGD motif and investigated their pharmacokinetics and utility for integrin αvβ3 imaging in tumors. The MccJ25 variant with an RGD motif in the loop region and a lysine substitution at the C-terminus (MccJ25(RGDF)GtoK) was produced in E. coli transfected with plasmid DNA containing the MccJ25 biosynthetic gene cluster (mcjABCD). [64Cu]Cu-MccJ25(RGDF)GtoK was synthesized using the C-terminal lysine labeled with copper-64 (t1/2 = 12.7 h) via a bifunctional chelator; it showed stability in 90% mouse plasma for 45 min. Using PET imaging for integrin αvβ3 positive U87MG tumor bearing mice, [64Cu]Cu-MccJ25(RGDF)GtoK could clearly distinguish the tumor, and its accumulation was significantly higher than that of MccJ25(GIGT)GtoK without the binding motif for integrin αvβ3. Furthermore, MccJ25(RGDF)GtoK enabled visualization of only U87MG tumors but not MCF-7 tumors with low integrin αvβ3 expression in double tumor-bearing mice. In ex vivo biodistribution analysis, the integrin αvβ3 non-specific accumulation of [64Cu]Cu-MccJ25(RGDF)GtoK was significantly lower in various tissues, except for the kidneys, as compared to the control probe ([64Cu]Cu-cyclic RGD peptide). These results of the present study indicate that 64Cu-labeling methods are appropriate for the synthesis of MccJ25-based PET probes, and [64Cu]Cu-MccJ25 variants are useful tools for cancer molecular imaging

Structural and immunostimulatory properties of Y-shaped DNA consisting of phosphodiester and phosphorothioate oligodeoxynucleotides.

Y-shape formation increased the immunostimulatory activity of phosphodiester (PO) oligodeoxynucleotides (ODNs) containing CpG motif. In this study, PO CpG ODN or CpG ODN containing nuclease-resistant phosphorothioate (PS) linkages, i.e., PS CpG ODN or PO CpG ODN with three PS linkages at the both ends (PS3), was mixed with two PO- or PS ODNs to prepare Y-shaped DNA (Y-DNA) containing a potent CpG motif. The melting temperature of Y-DNA decreased with increasing number of PS linkages. Y(PS/PO/PO), which contained PS CpG ODN, showed the greatest activity to induce tumor necrosis factor-α release from macrophage-like RAW264.7 cells, followed by Y(PS3/PO/PO). However, the high activity of Y(PS/PO/PO) was due to that of PS CpG ODN, and Y-shape formation had no significant effect on the activity. Furthermore, PS CpG ODN of Y(PS/PO/PO) was efficiently taken up by cells, but other PO ODNs in the Y-DNA were not, indicating that PS CpG ODN in Y-DNA behave like single stranded PS CpG ODN. In quite contrast, the immunostimulatory activity of PS3 CpG ODN was significantly increased by Y-shape formation. In conclusion, Y-shape formation and PS substitution can be used simultaneously to increase the immunostimulatory activity of CpG ODN, but extensive substitution should be avoided because it diminishes the benefits of Y-shape formation

Efficient delivery of immunostimulatory DNA to mouse and human immune cells through the construction of polypod-like structured DNA.

Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator

Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice.

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells

Injectable, self-gelling, biodegradable, and immunomodulatory DNA hydrogel for antigen delivery.

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

Optimal Arrangement of Four Short DNA Strands for Delivery of Immunostimulatory Nucleic Acids to Immune Cells.

Nanosized DNA assemblies are useful for delivering immunostimulatory cytosine-phosphate-guanine (CpG) DNA to immune cells, but little is known about the optimal structure for such delivery. In this study, we designed three different DNA nanostructures using four 55-mer oligodeoxynucleotides (ODNs), that is, tetrapod-like structured DNA (tetrapodna), tetrahedral DNA (tetrahedron), and tetragonal DNA (tetragon), and compared their potencies. Electrophoresis showed that tetrapodna was obtained with high yield and purity, whereas tetrahedron formed multimers at high ODN concentrations. Atomic force microscopy revealed that all preparations were properly constructed under optimal conditions. The thermal stability of tetrapodna was higher than those of the others. Dynamic light scattering analysis showed that all of the assemblies were about 8 nm in diameter. Upon addition to mouse macrophage-like RAW264.7 cells, tetrahedron was most efficiently taken up by the cells. Then, a CpG DNA, a ligand for toll-like receptor 9, was linked to these DNA nanostructures and added to RAW264.7 cells. CpG tetrahedron induced the largest amount of tumor necrosis factor-α, followed by CpG tetrapodna. Similar results were obtained using human peripheral blood mononuclear cells. Taken together, these results indicate that tetrapodna is the best assembly with the highest yield and high immunostimulatory activity, and tetrahedron can be another useful assembly for cellular delivery if its preparation yield is improved

DNA nanotechnology-based development of delivery systems for bioactive compounds.

Nucleic acids, DNA and RNA, not only allow transfer and replication of densely coded genetic information, but also act as danger signals triggering innate immune response. Recent progress in the design and construction of nano-sized structures using DNA has opened a new field of nanotechnology. The unique properties of nano-sized DNA constructs can be exploited to develop programmable materials for efficient delivery of bioactive compounds. In this review, recent advances in DNA nanotechnology and its applications as delivery systems are summarized. In particular, we focus on the delivery of DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotide, or CpG motif, to immune cells expressing Toll-like receptor 9. Recent studies have shown that precisely designed DNA constructs, such as multi-branched DNA, polyhedral DNA, and DNA origami, can be used to enhance the biological activity of CpG DNA

Self-Assembling DNA Dendrimer for Effective Delivery of Immunostimulatory CpG DNA to Immune Cells

DNA dendrimers consisting of several branched DNA units connected to each other using DNA ligase were quite effective for the delivery of immunostimulatory CpG DNA to immune cells. Therapeutic application of such DNA dendrimers, however, is hampered by the use of the ligase. Here, we report that self-assembling DNA dendrimers with high immunostimulatory potency can be prepared without DNA ligases. Annealing of DNA consisting of DNA units with elongated adhesive ends resulted in the formation of DNA dendrimers. Atomic force microscopy revealed that the several preparations of DNA dendrimers resulted in dendritic structures as designed. The cellular uptake of DNA dendrimers by mouse macrophage-like RAW264.7 cells and subsequent release of tumor necrosis factor-α were dependent on the structural complexity of the dendrimers. These results indicate that the ligation-free, self-assembling DNA dendrimers are a potent system for the delivery of immunostimulatory CpG DNA to immune cells