Toxic and teratogenic silica nanowires in developing vertebrate embryos

Abstract

Silica-based nanomaterials show promise for biomedical applications such as cell-selective drug delivery and bioimaging. They are easily functionalized, which allows for the conjugation or encapsulation of important biomolecules. Although recent in vitro studies suggested that silica-derived nanomaterials are nontoxic, in vivo studies of silica nanomaterial toxicity have not been performed. Using the embryonic zebrafish as a model system, we show that silica nanomaterials with aspect ratios greater than 1 are highly toxic (LD(50) = 110 pg/g embryo) and cause embryo deformities, whereas silica nanomaterials with an aspect ratio of 1 are neither toxic nor teratogenic at the same concentrations. Silica nanowires also interfere with neurulation and disrupt expression of sonic hedgehog, which encodes a key midline signaling factor. Our results demonstrate the need for further testing of nanomaterials before they can be used as platforms for drug delivery.

From the clinical editor: Silica-based nanomaterials show promise for biomedical applications such as cell-selective drug delivery and bioimaging. Using an embryonic zebrafish model system silica nanomaterials with aspect ratios greater than one were found to be highly toxic; whereas silica nanomaterials with an aspect ratio of one are neither toxic nor teratogenic. These results demonstrate the need for testing "nanomaterials" before they can be used as platforms for drug delivery.

Figure 1

Silica nanomaterials retain fuorophore after sonication and incubation in cell-free solutions. (A) FITC-conjugated silica nanowires retain external fuorophore conjugate after sonication and incubation in a solution designed to mimic intracellular conditions. (B) Supernatant showing the lack of FITC departure from the nanowire substrate. (C) Rhodamine-doped silica nanoparticles do not leach fluorescent material after incubation in a solution designed to mimic intracellular conditions. (D) Supernatant showing the lack of fluorescent material. Scale bar in (A) (applies to all) = 20 µm.

Figure 2

Silica nanomaterials show material-specific effects on survival of zebrafish embryos. (A) Unmodified silica nanowires show dose-dependent toxicity to embryos, predominately between 8 and 20 hours postfertilization (hpf). (B) Unmodified silica nanoparticles have negligible effects on embryo survival. Standard deviations (from clutch to clutch) for these experiments ranged from 0.004 to 0.013 and were omitted for figure clarity. (C) Dose-response curves for silica nanowires and silica nanoparticles. The LD₅₀s were 110 pg/g embryo for silica nanowires and 10 ng/g embryo for silica nanoparticles.

Figure 3

Functionally modified silica nanomaterials show material-specific effects on survival of zebrafish embryos. (A) FITC-conjugated nanowires show dose-dependent toxicity to embryos, predominately between 8 and 20 hpf. (B–D) Silica nanoparticles modified with (B) amine groups, (C) FITC, or (D) rhodamine have negligible effects on embryo survival. Standard deviations (from clutch to clutch) for these experiments ranged from 0.006 to 0.09 and were omitted for figure clarity.

Figure 4

Toxicity of silica nanomaterials is material- and exposure time-dependent. (A–F) Embryos were exposed at 36 hpf via microinjection into the yolk to (A) unmodified silica nanowires, (B) FITC-modified silica nanowires, (B) unmodified silica nanoparticles, (D) amine-modified silica nanoparticles, (E) FITC-modified silica nanoparticles, or (F) rhodamine-modified silica nanoparticles, and mortality was assessed at 132 hpf. No tested material was appreciably toxic using this exposure regime. (G, H) Embryos were exposed at 6 hpf via microinjection into the yolk to (G) FITC-modified silica nanowires or (H) rhodamine-modified silica nanoparticles, and mortality was assessed at 132 hpf. Nanowires but not nanoparticles are toxic to developing zebrafish embryos using this exposure regime. Standard deviations (from clutch to clutch) for these experiments ranged from 0.035 to 0.071 and were omitted for figure clarity.

Figure 5

Exposure to silica nanowires has teratogenic effects in zebrafish embryos. (A–C) Nanowire-exposed (at 0 hpf or 6 hpf) embryos with gross deformities (assessed at 60 hpf), such as (A) holoprosencephaly with cyclopia, (B) holoprosencephaly with anophthalmia, and (C) anterior axis duplication. (D) Control (water-injected) embryo assessed at 60 hpf. Scale bar in (D) (applies to all) = 50 µm. (e) eye, (h) heart, (y) yolk.

Figure 6

Modified silica nanowires are distributed throughout the embryo after exposure via microinjection into the yolk. (A) Bright-field image of embryo exposed at 6 hpf and imaged at 8 hpf(Y, yolk; ES, embryonic shield). (B) Same embryo imaged with epifluorescence optics showing the distribution of FITC-conjugated nanowires. (C) Merged image. Scale bar in (A) (applies to all) = 50 µm.

Figure 7

Modified silica nanoparticles are distributed throughout the embryo after exposure via microinjection into the yolk. (A–C) Embryo exposed to rhodamine-conjugated silica nanoparticles at 6 hpf and imaged at 8 hpf (Y, yolk; ES, embryonic shield); (A) bright-field, (B) epifluorescence, and (C) merged images show nanomaterial distribution. (D–F) Embryo exposed to rhodamine-conjugated silica nanoparticles at 36 hpf and imaged at 38 hpf; (D) bright-field, (E) epifluorescence, and (F) merged images show distribution of nanomaterials within the head and eye (arrow in F). Scale bars in (A) (applies to A–C) and (D) (applies to D–F) = 100 µm.

Figure 8

Silica nanowire exposure disrupts sonic hedgehog (shh) expression and neurulation. (A) Control, shh2.2:GFP transgenic embryo injected with water at 0 hpf and imaged at 14 hpf. (B) Transgenic embryo exposed to unmodified silica nanowires at 0 hpf and imaged at 14 hpf, showing abnormal neural keel morphology (arrows) and reduced expression of sonic hedgehog (arrowhead). Scale bar in (B) (applies to both) = 50 µm. Embryos were imaged using a combination of bright-field and epifluorescence microscopy.