Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
, 10, 1179670717694523
eCollection

In Vitro Neurotoxicity Resulting From Exposure of Cultured Neural Cells to Several Types of Nanoparticles

Affiliations
Review

In Vitro Neurotoxicity Resulting From Exposure of Cultured Neural Cells to Several Types of Nanoparticles

Stephen F Larner et al. J Cell Death.

Abstract

Laboratory and industrial production of various nanoparticles, single-walled nanotubes (SWNTs), fullerene (C60), cadmium selenide (CdSe) quantum dots, carbon black (CB), and dye-doped silica nanospheres (NSs), has greatly increased in the past 15 years. However, little research has been done to analyze the toxicity of these materials. With recent studies showing that nano-substances can cross the blood-brain barrier, we examined the neurotoxicity of these manufactured nanoparticles. By employing the rat PC-12 neuronal-like cell line as the basis for our studies, we were able to evaluate the toxicity caused by these five nanoparticles. The level of toxicity was measured by testing for cell viability using the lactate dehydrogenase (LDH) cell viability assay, morphological analysis of changes in cellular structures, and Western blot analyses of αII-spectrin breakdown products (SBDP) as cell death indicators. Our results showed cytotoxicity in nondifferentiated PC-12 cells exposed to CB (10-100 μg/mL), SWNTs (10-100 μg/mL), C60 (100 μg/mL), CdSe (10 μg/mL), CB (500 μg/mL), and dye-doped silicon NSs (10 μg/mL). Exposure to higher concentrations (100 μg/mL) of SWNTs, CB, and C60 increased the formation of SBDP150/145, as well as cell membrane contraction and the formation of cytosolic vacuoles. The incorporations of the nanoparticles into cell cytoplasm were observed using the fluorescent dye-doped NSs in both nondifferentiated and nerve growth factor (NGF)-differentiated PC-12 cells. When PC-12 cells are differentiated, they appeared to be even more sensitive to cytotoxicity of nanoparticles such as CB 10 nm (10-100 μg/mL), CB 100 nm (10-100 μg/mL), and CdSe (1-10 μg/mL).

Keywords: Nanoparticles; calpain; cell death; neurotoxicity.

Conflict of interest statement

DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Various nanoparticles causing cytotoxicity in nondifferentiated rat PC-12 cells, as measured by LDH release. LDH levels after 24-hour incubation with carbon black Z (5 nm), nanotubes, fullerenes (C60), cadmium selenide (CdSe), and silica nanospheres (doped with Green dye = Oregon Green 488, or Red dye = tetramethylrhodamine). Those that are significantly higher than control are indicated (Student’s t-test, **P < 0.01 or *P < 0.05; N = 6).
Figure 2.
Figure 2.
Rat nondifferentiated PC-12 cells morphology after exposure to various nanoparticles for 24 hours. (A) Control nondifferentiated PC-12 cells, (B) fullerenes (C60), 100 μg/mL, (C) carbon black, 100 μg/mL, and (D) single-walled nanotubes (SWNTs), 100 μg/mL. Yellow arrows indicate vacuole formation in NP-treated cells. Scale bar = 50 μm.
Figure 3.
Figure 3.
αII-spectrin and SBDP patterns in various nanoparticle exposures to nondifferentiated PC-12 cells. (A) Representative immunoblot for αII-spectrin. (B) Quantification of intact αII-spectrin reduction and increased SBDP145. Representative of three blots (Student’s t-test, **P < 0.01 or *P < 0.05; N = 3).
Figure 4.
Figure 4.
Fluorescence images of nondifferentiated PC-12 cells exposed to Oregon Green 488 dye-doped silica nanospheres (10 μg/mL) for 24 hours. (A) Phase-contrast microscopy, (B) fluorescence microscopy (green), (C) DAPI nuclear stain (blue), and (D) merged image of NP fluorescence and DAPI. Yellow arrows indicate vacuole formation in NP-treated cells. Scale bar = 50 μm.
Figure 5.
Figure 5.
Incorporation of fluorescent dye-doped silica nanospheres into cytoplasm of nondifferentiated PC-12 cells. (A) Combined phase-contrast and fluorescence microscopy (Excitation 340 nm, Emission 460 nm). Oregon Green 488 doped silica (10 μg/mL) found throughout the cytoplasm and perinuclear zone (green spots, shown with yellow arrows). (B) Corresponding phase contrast with nuclear DAPI staining showing location of cell nuclei (white arrow). Scale bar = 50 μm.
Figure 6.
Figure 6.
Cytotoxic effects of various nanoparticles on NGF-differentiated PC-12 cells. LDH release levels after 24-hour incubation with carbon black (5 and 100 nm diameter) and cadmium selenide NPs were significantly higher (at both 1 and 10 μg/mL) than control NGF-differentiated PC-12 cells (Student’s t-test, *P < 0.01).
Figure 7.
Figure 7.
Effects of CdSe on NGF-differentiated PC-12 cells morphology. (A) Differentiated PC-12 cells that show fine and long processes (red arrows). (B) Cadmium selenide NP (10 μg/ml) treated cells have shorten and abnormal processes (yellow arrows). (C) Precess length comparison of the control and CdSe-treated differentiated PC-12 cells. Scale bar = 50 μm.
Figure 8.
Figure 8.
NGF-differentiated PC-12 TMR-dye-doped nanospheres (10 μg/mL) for 24 hours. (A, C) Phase contrast overlaid with fluorescent image of TMR-NS (red) and nuclear dye DAPI (blue). (B, D) Phase-contrast microscopy alone (scale bar = 20 μm). (C, D) Higher magnification shows that NP has perinuclear localization (yellow arrows).

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

References

    1. Dang Y, Zhang Y, Fan L, Chen H, Roco MC. Trends in worldwide nanotechnology patent applications: 1991 to 2008. J Nanopart Res. 2010;12(3):687–706. - PMC - PubMed
    1. Wiesner MR, Lowry GV, Casman E, et al. Meditations on the ubiquity and mutability of nano-sized materials in the environment. ACS Nano. 2011;5(11):8466–8470. - PubMed
    1. Bobo D, Robinson KJ, Islam J, Thurecht KJ, Corrie SR. Nanoparticle-based medicines: a review of FDA-approved materials and clinical trials to date. Pharm Res. 2016;33(10):2373–2387. - PubMed
    1. Leonard F, Curtis LT, Yesantharao P, et al. Enhanced performance of macrophage-encapsulated nanoparticle albumin-bound-paclitaxel in hypo-perfused cancer lesions. Nanoscale. 2016;8(25):12544–12552. - PMC - PubMed
    1. Conde J, Larguinho M, Cordeiro A, et al. Gold-nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis. Nanotoxicology. 2014;8(5):521–532. - PubMed

LinkOut - more resources

Feedback