Research ArticleNANOMATERIALS

Cellular uptake and dynamics of unlabeled freestanding silicon nanowires

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Science Advances  16 Dec 2016:
Vol. 2, no. 12, e1601039
DOI: 10.1126/sciadv.1601039
  • Fig. 1 SiNW internalization.

    (A) Schematic illustration of SiNW internalization. (B) Confocal fluorescence micrograph of HUVECs (actin, red; tubulin, green) demonstrating SiNW internalization (blue scattering). Maximum projection in the xy plane (left; scale bar, 10 μm), interpolated projection in the yz plane (middle; height, 3.5 μm), and a thin confocal section taken along the dashed line segment n (right; height, 3.5 μm; length, 48.3 μm). (C) SEM micrograph of a HUVEC containing a SiNW [scale bar (top), 10 μm]. The magnified highlighted region indicates that the SiNW is embedded under the cell’s membrane [scale bar (bottom), 300 nm]. (D) TEM micrograph of a HUVEC thin section (~250 nm thick), with higher magnification insets, illustrating the distribution of internalized wires, both in vesicles and in the cytosol [scale bars, 1 μm (top) and 200 nm (insets)].

  • Fig. 2 Active SiNW transport.

    (A) SEPC micrograph of a SiNW before (top) and during (bottom; t = ~7 min per frame) internalization (scale bar, 15 μm), with tips 1 and 2 indicated by red and blue markers, respectively. (B) Path of travel for both tips of the SiNW as a function of time. (C) Instantaneous velocity of the SiNW before (15-frame interval) (I), during (II), and after (III) active transport, with the corresponding rolling MSD diffusivity exponent α, indicating an active transport process. The diffusivity exponent α was obtained over a rolling 30-frame period. All values given are for tip 1 (red).

  • Fig. 3 Ensemble SiNW internalization dynamics using SiNW-cell overlap.

    (A) Corresponding PC (top) and DF (bottom) micrographs taken at 2 hours (top set) and 20 hours (bottom set) after HUVEC incubation with SiNWs (scale bars, 25 μm), indicating increased SiNW-cell overlap and clustering in the perinuclear region (artificial cell outline highlighted in teal). (B) Uptake statistics of SiNWs with varying growth lengths (HUVECs) (top) and in multiple cell lines (bottom) after 24 hours, showing that longer wires are more likely to be internalized on the basis of geometric considerations and that cardiomyocytes (Cardio) and DRG neurons (Neuron) do not internalize unmodified SiNWs, whereas J774A.1 macrophages (Macro) show larger uptake rates. Average SiNW length by growth time: 10 min: 9.8 μm; 20 min: 14.5 μm; 30 min: 23.1 μm; 40 min: 31.7 μm. (C) Example ensemble SiNW-cell overlap (black dots) and cell confluence (red dots) trace as a function of time for unmodified SiNWs in HUVECs. Larger-than-confluence overlap indicates SiNW internalization. Expected overlap trend (black line) fit using the 2D random walker model (Dt = 410 μm2/hour, R2 = 0.93). Cell confluence modeled as an exponential fit (red line).

  • Fig. 4 Mechanistic and morphological studies.

    (A) Positive control study of Cyto D (actin inhibitor) showing the SiNW-cell overlap (black dots), cell confluence (red dots), and the expected overlap trend (black line), modeled on the first 8 hours of internalization (internal control) before drug introduction (red arrow). Cell confluence modeled as an exponential fit (red line). (B) Endocytosis inhibitors: dynasore (Dynamin; left), chlorpromazine (Clathrin; middle), and nystatin (Lipid Rafts; right), indicating dynamin’s critical role in SiNW internalization. (C) SEM micrograph showing membrane extension along a SiNW (scale bar, 500 nm). (D) Time-lapse SEPC micrographs of a membrane extending along a SiNW before cellular uptake (left) (scale bar, 5 μm). Distance of the protrusion’s leading edge from the base of the SiNW over time (top right), with the corresponding instantaneous velocities (bottom right). Base membrane and nanowire tip distances given as solid and dotted lines, respectively. Velocities smoothed over an 11-frame interval. (E) TEM micrograph showing a long intercellular SiNW protruding from a vesicle into the cytosol (scale bar, 250 nm).

  • Fig. 5 A5 as a phagocytosis inhibitor of SiNWs.

    (A) Schematic illustration showing the inhibition mechanism of A5. Unlike inhibitors that target the cell directly, the nonspecific binding of A5 to the negatively charged SiNW surface can screen the SiNW from uptake. (B) A5 inhibitor study, showing a reduced nanowire-cell overlap (black dots) as compared to the expected internalization (black line) (red arrow indicates dosage time). (C) Fluorescent and DF micrographs showing the level of A5-Cy3 absorption onto SiNWs in the presence (+) and absence (−) of serum in PBS and M200 solutions, indicating that the ability of A5 to bind with SiNWs is restricted by the presence of serum proteins. (D) Example micrograph showing the same region in the PC (left), DF (middle), and Cy3 (right, artificial color) channels, indicating that SiNW surfaces modified with Cy3-A5 (1 and 2) are excluded from the cell, whereas the internal controls (3) are endocytosed (artificial cell outline, teal) (scale bar, 30 μm). (E) SiNW-cell overlap in HUVECs after 24 hours of SiNWs with nonspecifically bound A5-Cy3 (A5 NS) and surface-functionalized A5-Cy3 (A5 SF), relative to an external (Ext. C.) and internal (Int. C.) control of unmodified SiNWs (β given as a percentage of external control) (**P < 0.01). DF and Cy3 were background-subtracted using a Gaussian spatial filter uniformly applied to each image.

  • Fig. 6 Schematic overview of SiNW internalization.

    After first coming into contact with the SiNW, the cell membrane extends along the entire length of the SiNW, engulfing the particle. This results in either complete or partial encapsulation of the SiNW into a small vesicle. The SiNW is then transported to the perinuclear region for processing. Eventually, the particle, through a yet unknown process, is able to leave the lysosome and is released to the cytosol.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/12/e1601039/DC1

    Supplementary Text

    table S1. Summary of inhibitor action on SiNW internalization.

    fig. S1. Perinuclear SiNW clustering.

    fig. S2. SiNW tracking algorithm.

    fig. S3. Stage drift controls.

    fig. S4. Linear SiNW transport trajectories.

    fig. S5. Migration coupled active transport.

    fig. S6. Serum-free SiNW internalization.

    fig. S7. Example MSD calculation.

    fig. S8. Nocodazole-inhibited SiNW transport.

    fig. S9. Active tug-of-war SiNW transport.

    fig. S10. Lysosome tracking.

    fig. S11. SiNW-cell colocalization determination.

    fig. S12. Concentration-dependent metabolic activity assay.

    fig. S13. Cell line–specific SiNW-cell colocalization.

    fig. S14. Ensemble HUVEC control samples.

    fig. S15. Multiple internalization events.

    fig. S16. Chlorpromazine-positive control.

    fig. S17. High-concentration A5 study.

    fig. S18. Cytosolic SiNW protein sheath formation.

    fig. S19. Prestained A5 SiNWs.

    fig. S20. Lova inhibitor study.

    movie S1. Single-cell SiNW active transport.

    movie S2. Bidirectional active tug-of-war SiNW transport.

    movie S3. Macrophage internalization of SiNWs.

    movie S4. SiNW membrane engulfment.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • table S1. Summary of inhibitor action on SiNW internalization.
    • fig. S1. Perinuclear SiNW clustering.
    • fig. S2. SiNW tracking algorithm.
    • fig. S3. Stage drift controls.
    • fig. S4. Linear SiNW transport trajectories.
    • fig. S5. Migration coupled active transport.
    • fig. S6. Serum-free SiNW internalization.
    • fig. S7. Example MSD calculation.
    • fig. S8. Nocodazole-inhibited SiNW transport.
    • fig. S9. Active tug-of-war SiNW transport.
    • fig. S10. Lysosome tracking.
    • fig. S11. SiNW-cell colocalization determination.
    • fig. S12. Concentration-dependent metabolic activity assay.
    • fig. S13. Cell line–specific SiNW-cell colocalization.
    • fig. S14. Ensemble HUVEC control samples.
    • fig. S15. Multiple internalization events.
    • fig. S16. Chlorpromazine-positive control.
    • fig. S17. High-concentration A5 study.
    • fig. S18. Cytosolic SiNW protein sheath formation.
    • fig. S19. Prestained A5 SiNWs.
    • fig. S20. Lova inhibitor study.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mov format). Single-cell SiNW active transport.
    • movie S2 (.mov format). Bidirectional active tug-of-war SiNW transport.
    • movie S3 (.mov format). Macrophage internalization of SiNWs.
    • movie S4 (.mov format). SiNW membrane engulfment.

    Files in this Data Supplement:

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