Development of a Cytosolic DNA Sensor Agonist Using GALA Peptide-Conjugated DNA and Long Single-Stranded DNA.

Cytosolic DNA sensors (CDSs) recognize DNA molecules that are abnormally located in the cytosol, thus leading to the activation of the stimulator of interferon genes (STING) and the induction of type 1 interferon. In turn, type 1 interferon evokes defensive reactions against viral infections and activates the immune system; therefore, the use of agonists of CDSs as cancer therapeutics and vaccine adjuvants is expected. Double-stranded DNA molecules with dozens to thousands of bases derived from bacteria and viruses are agonists of CDSs. However, DNA is a water-soluble molecule with a high molecular weight, resulting in poor cellular uptake and endosomal escape. In contrast, long single-stranded DNA (lssDNA) obtained by rolling circle amplification is efficiently taken up and localized to endosomes. Here we constructed a CDS-targeting lssDNA via the facilitation of its intracellular transport from endosomes to the cytosol. An endosome-disrupting GALA peptide was used to deliver the lssDNA to the cytosol. A peptide-oligonucleotide conjugate (POC) was successfully obtained via the conjugation of the GALA peptide with an oligonucleotide complementary to the lssDNA. By hybridization of the POC to the complementary lssDNA (POC/lssDNA), the CDS-STING pathway in dendritic cells was efficiently stimulated. GALA peptide-conjugated DNA seems to be a helpful tool for the delivery of DNA to the cytosol.

Development of Enzymatic Depletion Methods for Preparation of Small Extracellular Vesicles with Long Blood-Circulation Half-Life.

PURPOSE: Phosphatidylserine (PS)-deficient small extracellular vesicle (sEV) subpopulations (PS(-) sEVs) circulate in blood for long periods; hence, they are expected to have therapeutic applications. However, limited production of PS(-) sEVs makes their application difficult. In this study, a method for the preparation of such populations using an enzymatic reaction was developed. METHODS: Bulk sEVs collected from a cell culture supernatant via ultracentrifugation were subjected to an enzymatic reaction using phosphatidylserine decarboxylase (PSD). The yield of PS(-) sEVs was estimated using magnetic beads that bind to PS(+) sEVs. Then, the physical properties and pharmacokinetics (PK) of the sEVs were evaluated. RESULTS: Enzymatic depletion of PS exposed on sEV surfaces using PSD increased the yield of PS(-) sEVs. PSD treatment hardly changed the physicochemical properties of PS(-) sEVs. Moreover, the serum concentration profile and PK parameters of the PS(-) sEVs derived from PSD-treated bulk sEVs indicated a long blood-circulation half-life. CONCLUSIONS: Treatment of sEVs with PSD successfully reduced surface PS levels and increased the amount of the PS(-) sEV subpopulation among bulk sEVs. This protocol of efficient preparation of PS(-) sEVs based on PSD treatment, as well as information on the basic PK, can be foundational for the therapeutic application of sEVs.

Enhanced Immunostimulatory Activity of Covalent DNA Dendrons.

The present study focused on the design and synthesis of covalent DNA dendrons bearing multivalent cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) that can stimulate the immune system through the activation of TLR9. These dendrons were synthesized using branching trebler phosphoramidite containing three identical protecting groups that enabled the simultaneous synthesis of multiple strands on a single molecule. Compared with linear ODNs, covalent DNA dendrons were found to be more resistant to nuclease degradation and were more efficiently taken up by macrophage-like RAW264.7 cells. Cellular uptake was suggested to be mediated by macrophage scavenger receptors. The covalent DNA dendrons composed of multivalent immunostimulatory branches enhanced the secretion of proinflammatory cytokines TNF-α and IL-6 from RAW264.7 cells, and 9-branched DNA dendrons showed the highest enhancement. Given their enhanced efficacy, we expect covalent DNA dendrons to be useful structures of oligonucleotide medicines.

Combined use of chemically modified nucleobases and nanostructured DNA for enhanced immunostimulatory activity of CpG oligodeoxynucleotide.

Oligodeoxynucleotide (ODN) containing a cytosine-phosphate-guanine (CpG) motif, or CpG ODN, is considered suitable for treating immune diseases, including allergies. Although the phosphorothioate modification is used to enhance the stability and immunostimulatory activity of CpG ODNs, it is associated with the risk of adverse effects. Construction of nanostructured DNA assemblies, such as tripod- and hexapod-like structured DNAs, tripodna and hexapodna, respectively, were also found to increase this activity. The chemical modification of nucleobases could be another approach for enhancing CpG ODN activity. Here, we examined whether chemically modified nucleobase substitutions can enhance CpG ODN activity by measuring tumor necrosis factor α (TNF-α) release after addition to murine macrophage-like RAW264.7 cells. First, the guanine at the 18th position of phosphodiester CpG 1668 was substituted with several chemically modified guanines, and then the various guanines were substituted. Among all tested substitutions, 15,18-thdG, in which two guanines outside the CpG motif were substituted with the 2-aminothieno[3,4-d]pyrimidine guanine mimic (thdG), was the most effective. Compared to 32P-CpG 1668, 32P-15,18-thdG was taken up more efficiently by the RAW264.7 cells. Then, 15,18-thdG was incorporated into tripodna and hexapodna. 15,18-thdG/tri- or hexapodna induced higher TNF-α release from the RAW264.7 cells than PO CpG 1668/tri- or hexapodna, respectively. These results indicate that the thdG substitution is a useful effective strategy for enhancing the immunostimulatory activity of CpG DNAs in both single stranded and DNA nanostructure forms.

Adiponectin Stimulates Exosome Release to Enhance Mesenchymal Stem-Cell-Driven Therapy of Heart Failure in Mice.

Mesenchymal stem/stromal cells (MSCs) are cultured adult stem cells that originally reside in virtually all tissues, and the gain of MSCs by transplantation has become the leading form of cell therapy in various diseases. However, there is limited knowledge on the alteration of its efficacy by factors in recipients. Here, we report that the cardioprotective properties of intravenously injected MSCs in a mouse model of pressure-overload heart failure largely depend on circulating adiponectin, an adipocyte-secreted factor. The injected MSCs exert their function through exosomes, extracellular vesicles of endosome origin. Adiponectin stimulated exosome biogenesis and secretion through binding to T-cadherin, a unique glycosylphosphatidylinositol-anchored cadherin, on MSCs. A pharmacological or adenovirus-mediated genetic increase in plasma adiponectin enhanced the therapeutic efficacy of MSCs. Our findings provide novel insights into the importance of adiponectin in mesenchymal-progenitor-mediated organ protections.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Critical contribution of macrophage scavenger receptor 1 to the uptake of nanostructured DNA by immune cells.

Despite the efficient uptake of polypod-like nanostructured DNA, or polypodna, by macrophage-like RAW264.7 and other immune cells, the detailed mechanism has not been fully elucidated. Our previous study using HEK-Blue hTLR9 cells showed that transfection of macrophage scavenger receptor 1 (MSR1) increased the uptake of tetrapod-like structured DNA. Here, we investigated the involvement of MSR1 in the structure-dependent uptake of polypodna. Transfection of MSR1 to HEK-Blue hTLR9 cells pod number-dependently increased the uptake of polypodna, and its knockout in RAW264.7 cells reduced the uptake and subsequent cytokine release. To examine the binding of DNA with MSR1, biotinylated DNA added to RAW264.7 cells was cross-linked with cell surface proteins. Then, MSR1 cross-linked with polypodna, but not with single-stranded DNA. Similar results were obtained with murine primary immune cells. Taken together, MSR1 discriminates between simple and nanostructured DNAs and plays a dominant role in the efficient uptake of polypodna by immune cells.

Therapeutic Application of Small Extracellular Vesicles (sEVs): Pharmaceutical and Pharmacokinetic Challenges.

Small extracellular vesicles (sEVs), including exosomes as typical example, are cell-derived vesicles comprising lipid bilayer with a diameter approximately 100 nm. sEVs are endogenous delivery vehicles that deliver their contents such as nucleic acids and proteins to recipient cells. Because of their potential nature as endogenous delivery vehicles, therapeutic applications of sEVs as delivery systems of various drugs are expected. To develop sEV-based therapeutics, a variety of challenges should be overcome. In this review, we summarize the current status and future perspectives of therapeutic applications of sEVs. Several pharmaceutical and pharmacokinetic challenges will be discussed.

Enhanced Immunostimulatory Activity of CpG Oligodeoxynucleotide by the Combination of Mannose Modification and Incorporation into Nanostructured DNA.

The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5'-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.

Incorporation of Gelatin Microspheres into HepG2 Human Hepatocyte Spheroids for Functional Improvement through Improved Oxygen Supply to Spheroid Core.

The multicellular spheroid three-dimensional cell culture system can be used as a formulation for cell-based therapy. However, the viability and functions of the cells in the core region of the spheroid tend to decrease because of limited oxygen supply. In this study, we incorporated gelatin microspheres (GMS) into HepG2 human hepatocyte spheroids to allow oxygen to reach the spheroid core. GMS with an approximate diameter of 37 µm were fabricated by water-in-oil emulsification followed by freeze drying. GMS-containing HepG2 spheroids (GMS/HepG2 spheroids) were prepared by incubation of the cells with GMS at various mixing ratios in agarose gel-based microwells. Increasing the GMS ratio increased the diameter of the spheroids, and few spheroids formed with excess GMS. HepG2 cells in the GMS/HepG2 spheroids were more oxygenated than those in the GMS-free spheroids. GMS incorporation increased the viability of HepG2 cells in the spheroids and increased the CYP1A1 activity of the cells to metabolize 7-ethoxyresorufin, although mRNA expression of the CYP1A1 gene was hardly affected by GMS incorporation. These results indicate that incorporating GMS into HepG2 spheroids improves the hypoxic microenvironment in the spheroids and increases cell viability and CYP1A1 metabolic activity.

Development of RNA/DNA Hydrogel Targeting Toll-Like Receptor 7/8 for Sustained RNA Release and Potent Immune Activation.

Guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) is an agonist of Toll-like receptor (TLR) 7 and TLR8 and induces strong immune responses. A nanostructured GU-rich RNA/DNA assembly prepared using DNA nanotechnology can be used as an adjuvant capable of improving the biological stability of RNA and promoting efficient RNA delivery to target immune cells. To achieve a sustained supply of GU-rich RNA to immune cells, we developed a GU-rich RNA/DNA hydrogel (RDgel) using nanostructured GU-rich RNA/DNA assembly, from which GU-rich RNA can be released in a sustained manner. A hexapod-like GU-rich RNA/DNA nanostructure, or hexapodRD6, was designed using a 20-mer phosphorothioate-stabilized GU-rich RNA and six phosphodiester DNAs. Two sets of hexapodRD6 were mixed to obtain RDgel. Under serum-containing conditions, GU-rich RNA was gradually released from the RDgel. Fluorescently labeled GU-rich RNA was efficiently taken up by DC2.4 murine dendritic cells and induced a high level of tumor necrosis factor-α release from these cells when it was incorporated into RDgel. These results indicate that the RDgel constructed using DNA nanotechnology can be a useful adjuvant in cancer therapy with sustained RNA release and high immunostimulatory activity.

Effects of Localization of Antigen Proteins in Antigen-Loaded Exosomes on Efficiency of Antigen Presentation.

Exosomes are considered to be vehicles of antigen delivery. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of exosomes, might affect antigen presentation after exosomes are taken up by antigen-presenting cells; however, little is known about the importance of this phenomenon. In this study, lactadherin (LA) and group-specific antigen (Gag), exosome-tropic proteins that had previously been shown to cause the localization of luciferase to the outer surface and inner surface of exosomes, respectively [ Takahashi , Y. ; J. Biotechnol. 2013 ; Charoenviriyakul , C. ; Mol. Pharm. 2018 ], were used to examine the importance of the localization of antigen proteins in antigen presentation. Human embryonic kidney cells 293 (HEK293) were selected as exosomes producing cells. First, green fluorescent protein (GFP) was used to trace intracellular behavior of antigen proteins after uptake by murine dendritic DC2.4 cells. GFP-derived fluorescence signals were detected in cells only when GFP-inner-loaded (Gag-GFP) exosomes were added to them. Then, ovalbumin (OVA) was used as a model antigen protein, and OVA-loaded exosomes were added to bone marrow-derived dendritic cells. OVA-inner-loaded (Gag-OVA) exosomes showed enhanced class I antigen presentation capacity as compared with OVA-outer-loaded (OVA-LA) exosomes. Using PKH-labeled exosomes, it was found that the localization of OVA had very little effect on the cellular uptake of exosomes. These results indicate that the loading of antigen proteins inside exosomes helps in efficient antigen presentation.

Role of Extracellular Vesicle Surface Proteins in the Pharmacokinetics of Extracellular Vesicles.

Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin α6ß1, an integrin known to be involved in lung targeting, was degraded after ProK treatment. The ProK treatment significantly reduced the lung distribution of EVs after intravenous injection. These results indicate that the surface proteins of EVs such as integrin α6ß1 play some roles in pharmacokinetics in terms of reducing Vd and their distribution to the lung.

Possibility of Exosome-Based Therapeutics and Challenges in Production of Exosomes Eligible for Therapeutic Application.

Exosomes are cell-derived vesicles with a diameter 30-120 nm. Exosomes contain endogenous proteins and nucleic acids; delivery of these molecules to exosome-recipient cells causes biological effects. Exosomes derived from some types of cells such as mesenchymal stem cells and dendritic cells have therapeutic potential and may be biocompatible and efficient agents against various disorders such as organ injury. However, there are many challenges for the development of exosome-based therapeutics. In particular, producing exosomal formulations is the major barrier for therapeutic application because of their heterogeneity and low productivity. Development and optimization of producing methods, including methods for isolation and storage of exosome formulations, are required for realizing exosome-based therapeutics. In addition, improvement of therapeutic potential and delivery efficiency of exosomes are important for their therapeutic application. In this review, we summarize current knowledge about therapeutic application of exosomes and discuss some challenges in their successful use.

Enhanced Activity of Immunosuppressive Oligodeoxynucleotides by Incorporating Them into Hexapod-Like Nanostructured DNA.

A151 and other immunosuppressive oligodeoxynucleotides that act as Toll-like receptor (TLR) 9 antagonists are candidate agents for the treatment of autoimmune and inflammatory diseases in which TLR9 activation leads to harmful immune responses. Their efficient delivery to TLR9-positive target cells will increase their potency, but few attempts have been made to enhance their delivery. We previously reported that hexapod-like nanostructured DNA (hexapodna) enhanced the activity of immunostimulatory cytosine-phosphate-guanine (CpG) DNA by efficiently delivering it to immune cells. In this study, to enhance the immunosuppressive activity of A151, we designed a hexapodna containing six copies of the complementary sequence to A151. Structural analyses showed that A151-loaded hexapodna (supHexapodna) was obtained as designed. CpG 1668, which is a typical synthetic CpG DNA, induced tumour necrosis factor-α release from mouse macrophage-like RAW264.7 cells, and supHexapodna inhibited this more efficiently than A151. A flow cytometric analysis showed that the uptake of Alexa Fluor 488-labelled A151 by RAW264.7 cells significantly increased when it was incorporated into supHexapodna, whereas the uptake of Alexa Fluor 488-labelled CpG 1668 was hardly affected by A151 or supHexapodna. These results suggest that the hexapodna-mediated delivery of A151 can increase the potency of its TLR9-inhibitory activity towards immune cells.

Accelerated growth of B16BL6 tumor in mice through efficient uptake of their own exosomes by B16BL6 cells.

Exosomes are extracellular vesicles released by various cell types and play roles in cell-cell communication. Several studies indicate that cancer cell-derived exosomes play important pathophysiological roles in tumor progression. Biodistribution of cancer cell-derived exosomes in tumor tissue is an important factor for determining their role in tumor proliferation; however, limited studies have assessed the biodistribution of exosomes in tumor tissues. In the present study, we examined the effect of cancer-cell derived exosomes on tumor growth by analyzing their biodistribution. Murine melanoma B16BL6-derived exosomes increased the proliferation and inhibited the apoptosis of B16BL6 cells, which was associated with an increase and decrease in the levels of proliferation- and apoptosis-related proteins, respectively. GW4869-induced inhibition of exosome secretion decreased the proliferation of B16BL6 cells, and treatment of GW4869-treated cells with B16BL6-derived exosomes restored their proliferation. Next, we treated B16BL6 tumors in mice with B16BL6-derived exosomes and examined the biodistribution and cellular uptake of these exosomes. After the intratumoral injection of radiolabeled B16BL6-derived exosomes, most radioactivity was detected within the tumor tissues of mice. Fractionation of cells present in the tumor tissue showed that fluorescently labeled exosomes were mainly taken up by B16BL6 cells. Moreover, intratumoral injection of B16BL6-derived exosomes promoted tumor growth, whereas intratumoral injection of GW4869 suppressed tumor growth. These results indicate that B16BL6 cells secrete and take up their own exosomes to induce their proliferation and inhibit their apoptosis, which promotes tumor progression.

Amelioration of Experimental Autoimmune Encephalomyelitis in Mice by Interferon-Beta Gene Therapy, Using a Long-Term Expression Plasmid Vector.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Repeated injections of the interferon-ß (IFN-ß) protein are required for relapse prevention therapy in patients with MS. IFN-ß gene transfer can be an alternative treatment that continuously supplies IFN-ß protein to the patient without requiring repeated injections. In a previous study, we constructed a novel long-term IFN-ß-expressing plasmid vector (pMx-IFN-ß). In the present study, we examined whether gene transfer of pMx-IFN-ß could be effective for the treatment of MS in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Seven days after injection of the EAE-inducing peptide, the EAE mice received hydrodynamic injections pMx-IFN-ß. The severity of EAE symptoms in the pMx-IFN-ß-treated mice was significantly lower for 1 month than that observed in the untreated mice. An evaluation of blood-brain barrier (BBB) function, using Evans Blue, showed that injection of pMx-IFN-ß suppressed the BBB disruptions normally observed in EAE mice, while BBB disruptions remained evident in the untreated EAE mice. Histological analysis showed fewer invasive inflammatory cells in the spinal cords of the pMx-IFN-ß-treated mice than in the spinal cords of the other mice. Serum interferon gamma protein (IFN-γ) concentrations in the pMx-IFN-ß-treated mice were significantly lower than that in the untreated mice, indicating that IFN-ß gene transfer suppressed the production of IFN-γ from pathogenic T cells. These results indicate that IFN-ß transgene expression by single administration of the pMx-IFN-ß can be an effective long-term treatment for MS.

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.

Elucidation of the Mechanism of Increased Activity of Immunostimulatory DNA by the Formation of Polypod-like Structure.

PURPOSE: We previously demonstrated that the immunostimulatory activity of CpG DNA is increased by the formation of polypod-like structures. The present study was designed to elucidate the mechanism underlying this increase. METHODS: Tripodna (three pods) and hexapodna (six pods) were prepared. The cellular uptake of Alexa Fluor 488-labeled DNA samples was examined in several cell lines by measuring the MFI of cells. TNF-α release from RAW264.7 cells was measured after addition of polypodna containing CpG motifs. Dissociation of double stranded DNA was evaluated using FRET. RESULTS: Tripodna and hexapodna were efficiently taken up by macrophage-like RAW264.7 cells and dendritic DC2.4 cells, but not by fibroblast or endothelial cell lines. The uptake by RAW264.7 cells was highest for hexapodna, followed by tripodna, dsDNA, and ssDNA. The release of TNF-α from RAW264.7 cells was also highest for hexapodna. The ratio of TNF-α release to cellular uptake was highest for ssDNA, and lowest for dsDNA. Tripodna and hexapodna were more easily dissociated into single strands after cellular uptake than was dsDNA. CONCLUSIONS: The efficient cellular uptake and prompt dissociation into single strands can be directly related to the high immunostimulatory activity of polypod-like structured DNAs containing CpG motifs.

SELEX-Based Screening of Exosome-Tropic RNA.

Cell-derived nanosized vesicles or exosomes are expected to become delivery carriers for functional RNAs, such as small interfering RNA (siRNA). A method to efficiently load functional RNAs into exosomes is required for the development of exosome-based delivery carriers of functional RNAs. However, there is no method to find exosome-tropic exogenous RNA sequences. In this study, we used a systematic evolution of ligands by exponential enrichment (SELEX) method to screen exosome-tropic RNAs that can be used to load functional RNAs into exosomes by conjugation. Pooled single stranded 80-base RNAs, each of which contains a randomized 40-base sequence, were transfected into B16-BL6 murine melanoma cells and exosomes were collected from the cells. RNAs extracted from the exosomes were subjected to next round of SELEX. Cloning and sequencing of RNAs in SELEX-screened RNA pools showed that 29 of 56 clones had a typical RNA sequence. The sequence found by SELEX was enriched in exosomes after transfection to B16-BL6 cells. The results show that the SELEX-based method can be used for screening of exosome-tropic RNAs.