Research ArticleMOLECULAR BIOLOGY

Bottlebrush-architectured poly(ethylene glycol) as an efficient vector for RNA interference in vivo

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Science Advances  20 Feb 2019:
Vol. 5, no. 2, eaav9322
DOI: 10.1126/sciadv.aav9322
  • Fig. 1 Physical characterization of pacRNA.

    (A) Chemical structure of pacRNA. (B) A coarse-grained molecular dynamics simulation of the pacDNA (1-μs simulation with explicit water using the MARTINI force field). A crystal structure of Escherichia coli. RNase III is placed next to the pacRNA for size comparison. (C) Aqueous GPC chromatograms and agarose gel electrophoresis (1%; inset) of pacRNAs and free siRNA. (D) DLS intensity-average hydrodynamic diameter distribution of pacRNAClv. Inset, ζ potential measurements of siRNA and pacRNAs in Nanopure water. (E) TEM image of pacRNAClv, negatively stained with 2% uranyl acetate.

  • Fig. 2 Reductive release, hybridization, and enzymatic degradation of pacRNA.

    (A) Agarose gel electrophoresis showing the reductive release of siRNA from pacRNAClv in the presence of 10 mM DTT as a function of time; release profile by gel band densitometry analysis is shown on the right. (B) Schematics of enzymatic digestion kinetics assay based on fluorophore- and quencher-tagged RNA. (C and D) RNA hybridization and RNase III degradation kinetics for pacRNAs, a 40-kDa Y-shaped PEG-RNA conjugate, and free RNA.

  • Fig. 3 In vitro cellular uptake and gene silencing using pacRNA.

    (A) Flow cytometry measurement (total cell count: 10,000) of SKOV3 cells treated with PO RNA, PS RNA, and pacRNA (100 to 2000 nM) for 4 hours. (B) Mean fluorescence intensity of cells treated with free RNA (PS and PO) and pacRNAClv. a.u., arbitrary units. (C) Confocal microscopy images showing intracellular GSH-triggered release of siRNA from pacRNAClv in high-GSH cells (SKOV3 and SKBR3). Fluorescence is turned on when the fluorophore (fluorescein)–labeled siRNA is released from the quencher (dabcyl)–labeled brush polymer. Controls include low-GSH HDF cells (negative) and cells pretreated with 10 mM GSH-OEt (positive). (D) qRT-PCR measurement (mean ± SD; n = 3) of Bcl-2 transcript levels in SKOV3 cells treated with pacRNAs, free siRNA, and pacRNAClv containing a scrambled control sequence. (E) Bcl-2 protein levels characterized by Western blotting. (F) Cell apoptosis following sample treatment determined by annexin V and propidium iodide (PI) staining. Early apoptotic, late apoptotic, and necrotic cell populations (%) are shown in the lower right, upper right, and upper left quadrants, respectively. Results are representatives of three independent flow cytometry measurements. **P < 0.01 (two-tailed t test).

  • Fig. 4 Cytotoxicity and blood compatibility of pacRNA.

    (A) Cell viability of SKOV3 cells treated with pacRNAs and controls. (B) Hemolysis of human blood (type O+) treated with pacRNAs and controls, as determined by spectrophotometric measurement of hemoglobin present in the supernatant of centrifuged RBC suspensions. The %RBC hemolysis is defined as the percentage of hemoglobin present in the supernatant compared with the total hemoglobin released by Triton X-100 treatment. Inset, photograph of centrifuged RBC suspensions. (C) Activated partial thromboplastin times of plasma treated with pacRNA or ss PS RNA at equal RNA concentrations. ***P < 0.001 (two-tailed t test).

  • Fig. 5 Pharmacokinetics, biodistribution, efficacy, and safety assessment in vivo.

    (A) Plasma pharmacokinetics of PO RNA, PS RNA, pacRNA, and the brush polymer in C57BL/6 mice. (B) Fluorescence imaging of BALB/c nude mice bearing a human ovarian SKOV3 xenograft following intravenous injection of Cy5-labeled siRNA and pacRNAs, or the Cy5.5-labeled brush polymer. The red circle indicates the location of tumors. (C) Ex vivo imaging of tumors and other major organs 24 hours after injection. (D) Biodistribution determined by quantitative analysis of the fluorescence signals of ex vivo tumors and major organs. (E) Tumor volume changes in the course of 32 days with intravenous administration of PBS, pacRNAClv, and pacRNANClv at equivalent siRNA doses every fourth day (treatment started on day 0). (F) Body weight changes of tumor-bearing mice during the course of treatment. (G) Western blot of Bcl-2 in homogenized tumor tissues 96 hours after the last treatment. (H to J) Histological studies showing reduced Bcl-2 expression [immunohistochemistry staining (IHC)] (H), increased cell apoptosis (TUNEL) (I), and histologic apoptosis hallmarks (H&E staining) (J) in SKOV3 tumors treated with pacRNAClv compared with control groups. (K) Cytokine levels (TNF-α, IL-6, and IL-12) in the serum in C57BL/6 mice after 8 hours of treatment with pacRNAs and controls. **P < 0.01, ***P < 0.001 (two-tailed t test).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/2/eaav9322/DC1

    Supplementary Materials and Methods

    Scheme S1. Synthesis of pacRNA.

    Fig. S1. Synthetic scheme and characterization of dibenzocyclooctyne-modified RNA.

    Fig. S2. Additional characterization of brush polymers and pacRNAs.

    Fig. S3. Cellular uptake of PO siRNA, PS RNA, and pacRNA in SKOV3 cells.

    Fig. S4. Cellular uptake of PO siRNA, PS RNA (ss and ds), and pacRNA in SKBR3 cells.

    Fig. S5. Representative confocal images of SKBR3 cells treated with Cy3-labeled ss PS RNA or ds PS RNA for 4 h.

    Fig. S6. Bcl-2 down-regulation and cell apoptosis induced by pacRNA.

    Fig. S7. Fluorescence images of SKOV3 tumor cryosections following intravenous injections of siRNA, pacRNAs, and brush polymers.

    Fig. S8. Microscopic images of H&E-stained sections of various organs from mice after a 32-day treatment period with pacRNAs and PBS showing no apparent histological anomalies.

    Table S1. Oligonucleotide sequences.

    Table S2. Plasma pharmacokinetic parameters in C57BL/6 mice.

    References (4551)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Scheme S1. Synthesis of pacRNA.
    • Fig. S1. Synthetic scheme and characterization of dibenzocyclooctyne-modified RNA.
    • Fig. S2. Additional characterization of brush polymers and pacRNAs.
    • Fig. S3. Cellular uptake of PO siRNA, PS RNA, and pacRNA in SKOV3 cells.
    • Fig. S4. Cellular uptake of PO siRNA, PS RNA (ss and ds), and pacRNA in SKBR3 cells.
    • Fig. S5. Representative confocal images of SKBR3 cells treated with Cy3-labeled ss PS RNA or ds PS RNA for 4 h.
    • Fig. S6. Bcl-2 down-regulation and cell apoptosis induced by pacRNA.
    • Fig. S7. Fluorescence images of SKOV3 tumor cryosections following intravenous injections of siRNA, pacRNAs, and brush polymers.
    • Fig. S8. Microscopic images of H&E-stained sections of various organs from mice after a 32-day treatment period with pacRNAs and PBS showing no apparent histological anomalies.
    • Table S1. Oligonucleotide sequences.
    • Table S2. Plasma pharmacokinetic parameters in C57BL/6 mice.
    • References (4551)

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