Research ArticleGENETICS

Gold-DNA nanosunflowers for efficient gene silencing with controllable transformation

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Science Advances  02 Oct 2019:
Vol. 5, no. 10, eaaw6264
DOI: 10.1126/sciadv.aaw6264
  • Fig. 1 Scheme of self-assembled gold-DNA nanosunflowers for enhanced cellular uptake amount, tunable gene silencing efficacy, and controlled tumor inhibition effect by NIR irradiation.

    (A) (a) Assembly and disassembly of the large-sized nanostructure (200-nm gold-DNA nanosunflowers) from/to ultrasmall nanoparticles (2-nm Au-POY2T NPs). (b) Representative TEM image of the nanosunflowers. (c) Masterpiece: Sunflowers (Vincent van Gogh, 1889). (B) Left: In vivo tumor retention and penetration of transformable nanosunflowers. Right: Enhanced cellular uptake and controlled oncogene silencing process of the nanosunflowers in vitro. ① Large-sized nanosunflowers were taken up by an MCF-7 cell. ② The nanosunflowers standby in the cell cytoplasm. ③ Upon NIR irradiation, large-sized gold-DNA nanostructures dissociate and release small units (2-nm Au-POY2T NPs) to attack the cell nucleus. ④ The silencing sequence POY2T will bind to the P2 promoter of the c-myc oncogene and down-regulate the c-myc expression of MCF-7 cells, which can be controlled (ON/OFF) and regulated (Low/Medium/High) by the NIR irradiation.

  • Fig. 2 Morphology characterization of the self-assembled nanostructures (nanosunflowers).

    (A) TEM (200 kV) images of the nanosunflowers with enlarged structural details. (B) Bio-TEM (80 kV) images with enlarged polymer structural details. (C) High-resolution TEM (200 kV) images showing the distribution of ultrasmall NPs on the self-assembled nanostructure. (D) SEM images with enlarged surface topography of the nanosunflowers.

  • Fig. 3 Photothermal property and disassembly behavior study of the self-assembled nanostructures.

    (A) Visible absorption spectra of 2-nm core-sized NPs and 200-nm self-assembled nanostructures. a.u., absorbance unit. (B) Temperature response of self-assembled nanostructures, upon NIR irradiation, dispersed in water and cell culture medium. Mean values ± SD, n = 3. (C) Temperature rise of self-assembled nanostructures, upon NIR irradiation, dispersed in water and cell culture medium. (D) Change of maximum absorbance (767 nm) of 2-nm core-sized NPs and 200-nm self-assembled nanostructures upon NIR irradiation. (E and F) TEM observation of disassembly behavior of 200-nm self-assembled nanostructures before (top) and after (bottom) NIR irradiation (808 nm, 10 min). (G) Hydrodynamic diameter of (a) monodispersed 2-nm Au-POY2T NPs and size change of the 200-nm nanosunflowers before (b) and after (c and d) NIR irradiation for different time periods (3 and 10 min).

  • Fig. 4 Controlled nucleus localization and gene silencing study in vitro of the self-assembled nanostructures.

    (A) Schematic of the in vitro cell experimental setup for the controlled NP nucleus localization and gene regulation study. (B) Number of 2-nm Au-POY2T NPs localized in the MCF-7 cell nucleus with treatment of ① individual 2-nm Au-POY2T NPs, ② 200-nm nanosunflowers, and 200-nm nanosunflowers with NIR irradiation (10 min) after different preincubation times (③ 1, ④ 3, ⑤ 6, and ⑥ 12 hours). Mean values ± SD, n = 3. Statistical differences were determined by two-tailed Student’s t test; *P < 0.05 and **P < 0.01. (C) Confocal observation of distribution of fluorescein isothiocyanate–labeled nanosunflowers (green) before (top) and after (bottom) NIR irradiation in MCF-7 cells. Nucleus was labeled by 4′,6-diamidino-2-phenylindole (blue). (D) Bio-TEM image of the localization of large-sized nanosunflowers (top, red arrow) in the cytoplasm and distribution of released small NPs (bottom, blue arrow) in cytoplasm and nucleus after NIR irradiation in MCF-7 cells. (E) Cytotoxicity evaluation of MCF-7 cells with treatment of 200-nm nanosunflowers after NIR irradiation (after a period of preincubation time: 1, 3, 6, and 12 hours, respectively) compared to control, 2-nm Au-TIOP NPs, POY2T sequence, CA sequence, 2-nm Au-POY2T NPs, 200-nm nanosunflowers without NIR irradiation, and NIR exposure only. All the concentrations of treatments were at or equal to 1 μM in POY2T sequence and were tested after a total of 24 hours of incubation. Mean values ± SD, n = 3. Statistical differences were compared with the treatment group of ① individual 2-nm Au-POY2T NPs determined by two-tailed Student’s t test; *P < 0.05 and **P < 0.01. (F) C-myc mRNA level determined by real-time PCR after different treatments as described above. Mean values ± SD, n = 3. Statistical differences were determined by two-tailed Student’s t test; **P < 0.01 and ***P < 0.001. (G) C-myc protein levels determined by Western blot and (H) corresponding quantitative histogram after different treatments as described above. GAPDH, glyceraldehyde phosphate dehydrogenase.

  • Fig. 5 Controlled tumor growth inhibition study of the self-assembled nanostructures.

    (A) The MCF-7 tumor BALB/c nude mice model was established at day 0. After tumors were ready, the mice were randomly divided into nine groups and treated with 100 μl of various formulations (equivalent to 10 μM in POY2T sequence; group ① with 2-nm Au-POY2T NPs and groups ②, ③, ④, ⑤, and ⑥ with 200-nm nanosunflowers) at days 9, 12, and 15. In groups ③, ④, ⑤, and ⑥, the tumors were irradiated with a NIR laser for 10 min at 1, 3, 6, and 12 hours after each intravenous injection. Saline, NIR only, and POY2T were used as control groups. The (B) body weights and (C) tumor volumes were measured every 3 days. Scale bar, 1 cm. After the mice were sacrificed at day 24, all tumors were (D) isolated and (E) weighted, respectively. Mean values ± SD, n = 4. Statistical differences were determined by two-tailed Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001. (Photo credit: Ningqiang Gong, National Center for Nanoscience and Technology, China.) (F) Hematoxylin and eosin staining images of organs including the heart, liver, spleen, lung, kidney, and tumor after different treatments. Scale bar, 200 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaaw6264/DC1

    Fig. S1. Characterization of the as-synthesized NPs.

    Fig. S2. Cytotoxicity evaluation of MCF-7 cells with NIR irradiation at different times.

    Fig. S3. Stability test of self-assembled nanostructures.

    Fig. S4. Cellular uptake and intracellular distribution of NPs.

    Fig. S5. C-myc mRNA level determined in cytoplasm and nucleus separately by real-time PCR.

    Fig. S6. Safety evaluation of the nanosunflower structure.

    Fig. S7. Penetration behavior study of fluorescein isothiocyanate–labeled nanosunflowers in multicellular spheroid model.

    Fig. S8. Quantitative biodistribution of average Au content in tissues including the heart, liver, spleen, lung, kidney, and tumor after different treatments.

    Table S1. Hydrodynamic size distribution of Au-POY2T NPs and nanosunflowers before and after NIR irradiation for 3, 10, 12.5, or 15 min, respectively.

    Table S2. Histopathological scoring results of the H&E staining images.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Characterization of the as-synthesized NPs.
    • Fig. S2. Cytotoxicity evaluation of MCF-7 cells with NIR irradiation at different times.
    • Fig. S3. Stability test of self-assembled nanostructures.
    • Fig. S4. Cellular uptake and intracellular distribution of NPs.
    • Fig. S5. C-myc mRNA level determined in cytoplasm and nucleus separately by real-time PCR.
    • Fig. S6. Safety evaluation of the nanosunflower structure.
    • Fig. S7. Penetration behavior study of fluorescein isothiocyanate–labeled nanosunflowers in multicellular spheroid model.
    • Fig. S8. Quantitative biodistribution of average Au content in tissues including the heart, liver, spleen, lung, kidney, and tumor after different treatments.
    • Table S1. Hydrodynamic size distribution of Au-POY2T NPs and nanosunflowers before and after NIR irradiation for 3, 10, 12.5, or 15 min, respectively.
    • Table S2. Histopathological scoring results of the H&E staining images.

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