Enhanced Class I Tumor Antigen Presentation via Cytosolic Delivery of Exosomal Cargos by Tumor-Cell-Derived Exosomes Displaying a pH-Sensitive Fusogenic Peptide

Tumor-cell-derived exosomes contain endogenous tumor antigens and can be used as a potential cancer vaccine without requiring identification of the tumor-specific antigen. To elicit an effective antitumor effect, efficient tumor antigen presentation by MHC class I molecules on dendritic cells (DC) is desirable. Because DC endocytose exosomes, an endosomal escape mechanism is required for efficient MHC class I presentation of exosomal tumor antigens. In the present study, efficient cytosolic delivery of exosomal tumor antigens was performed using genetically engineered tumor-cell-derived exosomes and pH-sensitive fusogenic GALA peptide. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a streptavidin (SAV; a protein that binds to biotin with high affinity)–lactadherin (LA; an exosome-tropic protein) fusion protein to obtain SAV–LA-modified exosomes (SAV-exo). SAV-exo was mixed with biotinylated GALA to obtain GALA-modified exosomes (GALA-exo). Fluorescent microscopic observation using fluorescent-labeled GALA showed that the exosomes were modified with GALA. GALA-exo exerted a membrane-lytic activity under acidic conditions and efficiently delivered exosomal cargos to the cytosol. Moreover, DC treated with GALA-exo showed enhanced tumor antigen presentation capacity by MHC class I molecules. Thus, genetically engineered GALA-exo are effective in controlling the intracellular traffic of tumor-cell-derived exosomes and for enhancing tumor antigen presentation capacity

Construction of nanostructured DNA harbouring phosphorodiamidate morpholino oligonucleotide for controlled tissue distribution in mice

<p>Phosphorodiamidate morpholino oligonucleotides (PMOs) are a class of antisense oligonucleotides used in the treatment of neuromuscular diseases. Their major drawbacks are high blood clearance and poor cellular delivery. Previously, we demonstrated that tripod-like nanostructured DNA, or tripodna, was efficiently taken up by macrophages and dendritic cells. In this study, we used iodine-125(¹²⁵I)-labelled PMOs, designed a tripodna harbouring an ¹²⁵I-PMO (¹²⁵I-PMO/tripodna), and evaluated whether this tripodna could control the pharmacokinetic properties of PMO. Gel electrophoresis showed that ¹²⁵I-PMO was almost completely incorporated into the tripodna. Compared to ¹²⁵I-PMO, ¹²⁵I-PMO/tripodna was more efficiently taken up by macrophage-like RAW264.7 cells. Moreover, after intravenous injection into mice, the area under the plasma concentration–time curve of ¹²⁵I-PMO/tripodna was significantly larger than that of ¹²⁵I-PMO. The distribution of ¹²⁵I-PMO/tripodna in the liver and spleen at 24 h was 32- and 51-fold higher than that of ¹²⁵I-PMO, respectively. The fractionation of liver cells revealed that non-parenchymal cells were the major cells contributing to the hepatic uptake of ¹²⁵I-PMO/tripodna. These results indicate that tripodna has the potential to deliver PMO, particularly to the liver and spleen.</p

Near-Infrared Fluorescence Probes for Enzymes Based on Binding Affinity Modulation of Squarylium Dye Scaffold

We present a novel design strategy for near-infrared (NIR) fluorescence probes utilizing dye–protein interaction as a trigger for fluorescence enhancement. The design principle involves modification of a polymethine dye with cleavable functional groups that reduce the dye’s protein-binding affinity. When these functional groups are removed by specific interaction with the target enzymes, the dye’s protein affinity is restored, protein binding occurs, and the dye’s fluorescence is strongly enhanced. To validate this strategy, we first designed and synthesized an alkaline phosphatase (ALP) sensor by introducing phosphate into the squarylium dye scaffold; this sensor was able to detect ALP-labeled secondary antibodies in Western blotting analysis. Second, we synthesized a probe for β-galactosidase (widely used as a reporter of gene expression) by means of β-galactosyl substitution of the squarylium scaffold; this sensor was able to visualize β-galactosidase activity both in vitro and in vivo. Our strategy should be applicable to obtain NIR fluorescence probes for a wide range of target enzymes

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

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 (<i>R</i>_g) 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 <i>R</i>_g 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

Small-Molecule-Induced Clustering of Heparan Sulfate Promotes Cell Adhesion

Adhesamine is an organic small molecule that promotes adhesion and growth of cultured human cells by binding selectively to heparan sulfate on the cell surface. The present study combined chemical, physicochemical, and cell biological experiments, using adhesamine and its analogues, to examine the mechanism by which this dumbbell-shaped, non-peptidic molecule induces physiologically relevant cell adhesion. The results suggest that multiple adhesamine molecules cooperatively bind to heparan sulfate and induce its assembly, promoting clustering of heparan sulfate-bound syndecan-4 on the cell surface. A pilot study showed that adhesamine improved the viability and attachment of transplanted cells in mice. Further studies of adhesamine and other small molecules could lead to the design of assembly-inducing molecules for use in cell biology and cell therapy

Small-Molecule-Induced Clustering of Heparan Sulfate Promotes Cell Adhesion

Adhesamine is an organic small molecule that promotes adhesion and growth of cultured human cells by binding selectively to heparan sulfate on the cell surface. The present study combined chemical, physicochemical, and cell biological experiments, using adhesamine and its analogues, to examine the mechanism by which this dumbbell-shaped, non-peptidic molecule induces physiologically relevant cell adhesion. The results suggest that multiple adhesamine molecules cooperatively bind to heparan sulfate and induce its assembly, promoting clustering of heparan sulfate-bound syndecan-4 on the cell surface. A pilot study showed that adhesamine improved the viability and attachment of transplanted cells in mice. Further studies of adhesamine and other small molecules could lead to the design of assembly-inducing molecules for use in cell biology and cell therapy