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

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Abstract

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.

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Abbreviations

ANOVA:

One-way analysis of variance

BHQ:

Black hole quencher

CpG DNA:

DNA containing unmethylated cytosine-phosphate-guanine dinucleotides

DAPI:

4′,6-diamino-2-phenylindole

DS:

Dextran sulfate

EtBr:

Ethidium bromide

FRET:

Fluorescence resonance energy transfer

MFI:

Mean fluorescence intensity

ODN:

Oligodeoxynucleotide

PAGE:

Polyacrylamide gel electrophoresis

PBMC:

Human peripheral blood mononuclear cells

Polypodna:

Polypod-like structured DNA

SR-A:

Type A scavenger receptor

TLR9:

Toll-like receptor 9

Tm:

Melting temperature

TNF:

Tumor necrosis factor.

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Author information

Author notes

  1. Kohta Mohri

    Present address: Center Director’s Strategic Program, RIKEN Center for Life Science Technologies (CLST), 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan

Authors and Affiliations

  1. Laboratory of Drug Delivery System, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, 573-0101, Japan

    Kohta Mohri, Kengo Nagata, Shiori Toyama, Mao Nonomura & Shinji Sakuma

  2. Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan

    Shozo Ohtsuki, Yuki Takahashi, Yoshinobu Takakura & Makiya Nishikawa

  3. Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba, 278-8510, Japan

    Makiya Nishikawa

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  1. Kohta Mohri
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Correspondence to Kohta Mohri.

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Mohri, K., Nagata, K., Ohtsuki, S. et al. Elucidation of the Mechanism of Increased Activity of Immunostimulatory DNA by the Formation of Polypod-like Structure. Pharm Res 34, 2362–2370 (2017). https://doi.org/10.1007/s11095-017-2243-y

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  • DOI: https://doi.org/10.1007/s11095-017-2243-y

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