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Targeted gene silencing in vivo by platelet membrane–coated metal-organic framework nanoparticles

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Science Advances  27 Mar 2020:
Vol. 6, no. 13, eaaz6108
DOI: 10.1126/sciadv.aaz6108

Figures

  • Fig. 1 Platelet membrane–coated siRNA-loaded MOFs (P-MOF-siRNA) for gene silencing.

    To fabricate the P-MOF-siRNA formulation, siRNA-loaded MOF (MOF-siRNA) cores are generated by mixing the siRNA payload with Zn2+ and 2-methylimidazole (mim), followed by coating with natural cell membrane derived from platelets. When the P-MOF-siRNA nanoparticles are endocytosed by a target cell, the low pH of the endosomes causes escape of the siRNA into the cytosol. Upon incorporation with RNA-induced silencing complex (RISC), the target mRNA is then recognized and degraded, leading to gene silencing.

  • Fig. 2 Formulation and characterization.

    (A) Diameter of pristine MOF, MOF-siRNA, P-MOF, P-MOF-siRNA, and platelet (PLT) membrane vesicles after formulation (n = 3, mean + SD). (B) Zeta potential of pristine MOF, MOF-siRNA, P-MOF, P-MOF-siRNA, and platelet membrane vesicles after formulation (n = 3, mean + SD). (C) Transmission electron microscopy image of P-MOF-siRNA negatively stained with uranyl acetate (scale bar, 200 nm). (D) Encapsulation efficiency of siRNA inside P-MOF-siRNA at various siRNA inputs (n = 3, mean + SD). (E) Stability of MOF-siRNA and P-MOF-siRNA over time in PBS or serum-containing medium (n = 3, mean ± SD). (F) siRNA release from P-MOF-siRNA at pH 5.0 or pH 7.4 over time (n = 3, mean ± SD). (G and H) Degradation of siRNA, either in free form or in P-MOF-siRNA, when exposed to purified RNase (G) or serum-containing medium (H) for increasing amounts of time. (I) Western blots for three characteristic platelet surface markers (CD41, CD61, and P-selectin) in MOF-siRNA, platelet membrane vesicles, and P-MOF-siRNA. (J) Dot blot intensity of P-MOF-siRNA probed with antibodies against the intracellular or extracellular domains of CD47 (n = 3, mean + SD).

  • Fig. 3 In vitro cellular uptake and GFP knockdown.

    (A) Uptake of MOF-siRNA, P-MOF-siRNA, or R-MOF-siRNA by J774 macrophages after 24 hours of incubation (n = 3, mean + SD). (B and C) Secretion of proinflammatory cytokines IL-6 (B) or TNFα (C) by J774 macrophages after 24 hours of incubation with MOF-siRNA, P-MOF-siRNA, or R-MOF-siRNA (n = 3, mean + SD). *P < 0.05, **P < 0.01; NS, not significant; one-way analysis of variance (ANOVA). (D) Binding of fluorescently labeled P-MOF-siRNA, P-MOF-siRNA preblocked with anti–P-selectin, or R-MOF-siRNA to SK-BR-3 cells after incubation for 30 min (scale bar, 50 μm; nuclei, blue; nanoparticles, red). (E) Uptake of siRNA in SK-BR-3 cells 24 hours after incubation with free siRNA, P-MOF-siRNA, or R-MOF-siRNA. (F) Fluorescent visualization of siRNA localization in SK-BR-3 cells 1, 4, 8, and 24 hours after incubation with P-MOF-siRNA (scale bar, 20 μm; siRNA, green; nuclei, blue; endosomes, red). (G) Fluorescence of GFP-transduced SK-BR-3 cells after incubation with siRNAGFP, P-MOF, P-MOF-siRNANC, P-MOF-siRNAGFP, or R-MOF-siRNAGFP for 48 hours; wild-type (WT) cells were used to establish the baseline. (H) Visualization of gene knockdown in GFP-transduced SK-BR-3 cells after incubation with siRNAGFP, P-MOF, P-MOF-siRNANC, P-MOF-siRNAGFP, or R-MOF-siRNAGFP for 48 hours (scale bar, 200 μm; GFP, green).

  • Fig. 4 In vitro survivin knockdown.

    (A) Viability of SK-BR-3 cells after incubation with siRNASur, P-MOF, P-MOF-siRNANC, P-MOF-siRNASur, or R-MOF-siRNASur for 0, 24, 48, and 72 hours (n = 6, mean ± SD). (B) Western blot for survivin in SK-BR-3 cells after incubation with siRNASur, P-MOF, P-MOF-siRNANC, P-MOF-siRNASur, or R-MOF-siRNASur for 48 hours; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (C) Fluorescent visualization of survivin protein expression in SK-BR-3 cells after incubation with siRNASur, P-MOF, P-MOF-siRNANC, P-MOF-siRNASur, or R-MOF-siRNASur for 48 hours (scale bar, 20 μm; survivin, purple; nuclei, blue). (D) Relative survivin mRNA expression in SK-BR-3 cells after incubation with siRNASur, P-MOF, P-MOF-siRNANC, P-MOF-siRNASur, or R-MOF-siRNASur for 48 hours (n = 3, mean + SD).

  • Fig. 5 In vivo delivery and antitumor efficacy.

    (A) Nanoparticle biodistribution in the heart, liver, spleen, lungs, kidneys, and tumor 1 hour after intravenous administration with fluorescently labeled P-MOF-siRNASur or R-MOF-siRNASur (n = 3, mean + SD). a.u., arbitrary units. (B) Quantification of nanoparticle localization within the tumor 1 hour after intravenous administration with fluorescently labeled P-MOF-siRNASur or R-MOF-siRNASur (n = 3, mean + SD). (C) Ex vivo fluorescent imaging of tumors 1 hour after intravenous administration with fluorescently labeled P-MOF-siRNASur or R-MOF-siRNASur (H, high signal; L, low signal). (D) Growth kinetics of SK-BR-3 tumors implanted subcutaneously into nu/nu mice and treated intravenously with P-MOF-siRNASur or R-MOF-siRNASur every 3 days for a total of four administrations (n = 5; mean ± SEM). (E) Survival of the mice in (D) over time (n = 5). (F) Body weight of the mice in (D) over time (n = 5; mean ± SD).

  • Fig. 6 In vivo safety.

    H&E (A) and TUNEL (B) staining of histological sections from major organs, including the heart, liver, spleen, lungs, and kidneys, 24 hours after intravenous administration with a high dose of P-MOF-siRNASur (scale bars, 500 μm).

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