Original Article
Self-assembling DNA hydrogel-based delivery of immunoinhibitory nucleic acids to immune cells

https://doi.org/10.1016/j.nano.2015.08.004Get rights and content

Abstract

Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines.

From the Clinical Editor

Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.

Graphical Abstract

Takumi-shaped DNA consisting of two partially complementary immunoinhibitory oligodeoxynucleotides and Takumi-based DNA hydrogels were developed as a useful delivery vehicle for the oligodeoxynucleotides to immune cells for the inhibition of Toll-like receptor 9-mediated hyperinduction of proinflammatory cytokines.

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Section snippets

Chemicals

RPMI1640 medium was obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) was obtained from Hyclone (South Logan, UT, USA). Opti-modified Eagle’s medium (Opti-MEM) and MEM non-essential amino acids solution (NEAA) were purchased from Life Technologies (Carlsbad, CA, USA). Monothioglycerol was obtained from Wako Pure Chemical (Osaka, Japan). The 20-bp ladder was purchased from Takara Bio, Inc. (Shiga, Japan). Propidium iodide (PI) and blue dextran (average

Formation of Takumi and DNA hydrogel

Figure 1 shows the putative structures of the Takumi and Takumi-based DNA hydrogel. Two ODNs each were used to prepare the Takumi (iODN1 and iODN2) and DNA hydrogel (iODN1 and iODN3). The Takumi for the DNA hydrogel was designed such that both the 5′-ends and the 3′-ends are complementary to each other. Since these Takumi and DNA hydrogels contain INH-ODNs, we called them inhibitory Takumi (iTakumi) and inhibitory DNA hydrogel (iTakumiGel), respectively. Figure 2, A shows the PAGE analysis of

Discussion

Our previous studies demonstrated that the organization of CpG DNA into a branched form significantly increases its immunostimulatory activity.15, 16 In this study, we created a branched DNA, called Takumi, using two 33-mer ODNs, which was used as the main component of a DNA hydrogel. The AFM image clearly showed that the branched DNA adopted a Takumi shape as designed (Figure 2, B), which supports the notion that the smaller iTakumi units are also in a Takumi shape. An important finding of

Acknowledgment

The authors would like to thank Prof. Hiroshi Watanabe of Institute for Chemical Research, Kyoto University, for his helpful advice and discussion on the rheological properties of the samples.

References (30)

  • A. Krug et al.

    Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells

    Eur J Immunol

    (2001)
  • C. Schubert

    Boosting our best shot

    Nat Med

    (2009)
  • H. Yasuda et al.

    Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury

    Am J Physiol Renal Physiol

    (2008)
  • J. Tian et al.

    Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

    Nat Immunol

    (2007)
  • L.L. Stunz et al.

    Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells

    Eur J Immunol

    (2002)
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    The authors have no conflict of interest and no disclosures.

    This work was supported in part by a Grant-in-Aid for Scientific Research (B) (23390010) from the Japan Society for the Promotion of Science, by a Grant-in-Aid for Scientific Research on Innovative Areas “Carcinogenic spiral” (25114706) from theMinistry of Education, Culture, Sports, Science, and Technology of Japan, and by a Cross-Disciplinary Collaborative Research Promotion Project from the Institute for Integrated Cell-Material Sciences, Kyoto University.

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