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Nanodiamonds act as Trojan horse for intracellular delivery of metal ions to trigger cytotoxicity.

Zhu Y, Zhang Y, Shi G, Yang J, Zhang J, Li W, Li A, Tai R, Fang H, Fan C, Huang Q - Part Fibre Toxicol (2015)

Bottom Line: In addition, theoretical calculation and molecular dynamics (MD) computation were used to illustrate the adsorption properties of different metal ion on NDs as well as release profile of ion from ND-ion complexes at different pH values.Detailed investigation of ND-Cu2+ interaction showed that the amount of released Cu2+ from ND-Cu2+ complexes at acidic lysosomal conditions was much higher than that at neutral conditions, leading to the elevation of intracellular ROS level, which triggered cytotoxicity.The present experimental and theoretical results provide useful insight into understanding of cytotoxicity triggered by nanoparticle-ion interactions, and open new ways in the interpretation of nanotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Division of Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201800, China. zhuying@sinap.ac.cn.

ABSTRACT

Background: Nanomaterials hold great promise for applications in the delivery of various molecules with poor cell penetration, yet its potential for delivery of metal ions is rarely considered. Particularly, there is limited insight about the cytotoxicity triggered by nanoparticle-ion interactions. Oxidative stress is one of the major toxicological mechanisms for nanomaterials, and we propose that it may also contribute to nanoparticle-ion complexes induced cytotoxicity.

Methods: To explore the potential of nanodiamonds (NDs) as vehicles for metal ion delivery, we used a broad range of experimental techniques that aimed at getting a comprehensive assessment of cell responses after exposure of NDs, metal ions, or ND-ion mixture: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Trypan blue exclusion text, optical microscope observation, synchrotron-based scanning transmission X-ray microscopy (STXM) and micro X-ray fluorescence (μXRF) microscopy, inductively coupled plasma-mass spectrometry (ICP-MS), reactive oxygen species (ROS) assay and transmission electron microscopy (TEM) observation. In addition, theoretical calculation and molecular dynamics (MD) computation were used to illustrate the adsorption properties of different metal ion on NDs as well as release profile of ion from ND-ion complexes at different pH values.

Results: The adsorption capacity of NDs for different metal ions was different, and the adsorption for Cu2+ was the most strong among divalent metal ions. These different ND-ion complexes then had different cytotoxicity by influencing the subsequent cellular responses. Detailed investigation of ND-Cu2+ interaction showed that the amount of released Cu2+ from ND-Cu2+ complexes at acidic lysosomal conditions was much higher than that at neutral conditions, leading to the elevation of intracellular ROS level, which triggered cytotoxicity. By theoretical approaches, we demonstrated that the functional carbon surface and cluster structures of NDs made them good vehicles for metal ions delivery.

Conclusions: NDs played the Trojan horse role by allowing large amounts of metal ions accumulate into living cells followed by subsequent release of ions in the interior of cells, which then led to cytotoxicity. The present experimental and theoretical results provide useful insight into understanding of cytotoxicity triggered by nanoparticle-ion interactions, and open new ways in the interpretation of nanotoxicity.

No MeSH data available.


Related in: MedlinePlus

Interactions between NDs and Cu2+determine their internalization fate. a: STXM images of copper distribution in a typical control L929 cell (top left), cell after incubation with NDs (top right), Cu2+ (bottom left), and NDs-Cu2+ mixture (bottom right) for 24 h. The range of quantities noted by the color bar is from 3.2 × 10−6 to 7.0 × 10−6 in (top left), from 3.9 × 10−6 to 7.2 × 10−6 in (top right), from 2.4 × 10−6 to 7.2 × 10−6 in (bottom left) and from 6.5 × 10−6 to 5.0 × 10−5 in (bottom right). The scanning step was 50 nm. b: Imaging of intracellular copper distribution by microXRF. Elemental maps of copper (upper) and zinc (lower) are drawn. The size of a pixel is 3 μm × 3 μm. c: Intracellular Cu2+ concentration determined by ICP-MS (**p < 0.01, one-way ANOVA for comparison).
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Fig3: Interactions between NDs and Cu2+determine their internalization fate. a: STXM images of copper distribution in a typical control L929 cell (top left), cell after incubation with NDs (top right), Cu2+ (bottom left), and NDs-Cu2+ mixture (bottom right) for 24 h. The range of quantities noted by the color bar is from 3.2 × 10−6 to 7.0 × 10−6 in (top left), from 3.9 × 10−6 to 7.2 × 10−6 in (top right), from 2.4 × 10−6 to 7.2 × 10−6 in (bottom left) and from 6.5 × 10−6 to 5.0 × 10−5 in (bottom right). The scanning step was 50 nm. b: Imaging of intracellular copper distribution by microXRF. Elemental maps of copper (upper) and zinc (lower) are drawn. The size of a pixel is 3 μm × 3 μm. c: Intracellular Cu2+ concentration determined by ICP-MS (**p < 0.01, one-way ANOVA for comparison).

Mentions: Next, we used synchrotron-based scanning transmission X-ray microscopy (STXM) and micro X-ray fluorescence (μXRF) techniques to investigate the internalization fate of free Cu2+ and ND-Cu2+ mixture. IC50 value of Cu2+ (25 μg/mL) calculated from Additional file 1: Figure S2 was chosen for the succedent observations. From STXM images of a typical cell, we observed a significant increase in the amount of intracellular Cu2+ when exposed to the ND-Cu2+ mixture as compared with exposure to Cu2+ alone. (Figure 3a and Additional file 1: Figure S7). More importantly, it is found that for the NDs-Cu2+ exposure groups, large amount of Cu2+ inside the cells were mainly attached to NDs, illustrating that Cu2+ entered the cells in the form of ND-Cu2+ complexes. Another synchrotron-based μXRF experiment was performed to further examine the difference in intracellular Cu2+ concentration with or without NDs. Fluorescence spectra showed that cells cultured in medium containing 25 μg/mL Cu2+ yielded a ≈ 18 fold increase Cu Kа signal at 8.05 KeV compared with cells cultured in basal medium (see Additional file 1: Figure S8). Consistent with recent literature [34], we found that the total amount of Zn varied little in most of the samples and the Zn concentration followed the cell shape. Additionally, uptake of NDs-Cu2+ complex may influence Zn ion level inside cells due to toxicity. Elemental maps of a typical cell showed that the Cu2+ concentration in cells treated with ND-Cu2+ mixture was significantly higher than that in cells treated with Cu2+ alone, and no Cu2+ signal was detected in cells treated with NDs alone, which was consistent with that treated with basal medium (Figure 3b). Further transmission electron microscopy (TEM) observation as well as energy dispersive spectroscopy (EDS) analysis confirmed these X-ray imaging results (see Additional file 1: Figure S9). Moreover, both STXM and μXRF images showed that addition of NDs made the Cu-rich zone inside cells get more concentrated.Figure 3


Nanodiamonds act as Trojan horse for intracellular delivery of metal ions to trigger cytotoxicity.

Zhu Y, Zhang Y, Shi G, Yang J, Zhang J, Li W, Li A, Tai R, Fang H, Fan C, Huang Q - Part Fibre Toxicol (2015)

Interactions between NDs and Cu2+determine their internalization fate. a: STXM images of copper distribution in a typical control L929 cell (top left), cell after incubation with NDs (top right), Cu2+ (bottom left), and NDs-Cu2+ mixture (bottom right) for 24 h. The range of quantities noted by the color bar is from 3.2 × 10−6 to 7.0 × 10−6 in (top left), from 3.9 × 10−6 to 7.2 × 10−6 in (top right), from 2.4 × 10−6 to 7.2 × 10−6 in (bottom left) and from 6.5 × 10−6 to 5.0 × 10−5 in (bottom right). The scanning step was 50 nm. b: Imaging of intracellular copper distribution by microXRF. Elemental maps of copper (upper) and zinc (lower) are drawn. The size of a pixel is 3 μm × 3 μm. c: Intracellular Cu2+ concentration determined by ICP-MS (**p < 0.01, one-way ANOVA for comparison).
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Related In: Results  -  Collection

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Fig3: Interactions between NDs and Cu2+determine their internalization fate. a: STXM images of copper distribution in a typical control L929 cell (top left), cell after incubation with NDs (top right), Cu2+ (bottom left), and NDs-Cu2+ mixture (bottom right) for 24 h. The range of quantities noted by the color bar is from 3.2 × 10−6 to 7.0 × 10−6 in (top left), from 3.9 × 10−6 to 7.2 × 10−6 in (top right), from 2.4 × 10−6 to 7.2 × 10−6 in (bottom left) and from 6.5 × 10−6 to 5.0 × 10−5 in (bottom right). The scanning step was 50 nm. b: Imaging of intracellular copper distribution by microXRF. Elemental maps of copper (upper) and zinc (lower) are drawn. The size of a pixel is 3 μm × 3 μm. c: Intracellular Cu2+ concentration determined by ICP-MS (**p < 0.01, one-way ANOVA for comparison).
Mentions: Next, we used synchrotron-based scanning transmission X-ray microscopy (STXM) and micro X-ray fluorescence (μXRF) techniques to investigate the internalization fate of free Cu2+ and ND-Cu2+ mixture. IC50 value of Cu2+ (25 μg/mL) calculated from Additional file 1: Figure S2 was chosen for the succedent observations. From STXM images of a typical cell, we observed a significant increase in the amount of intracellular Cu2+ when exposed to the ND-Cu2+ mixture as compared with exposure to Cu2+ alone. (Figure 3a and Additional file 1: Figure S7). More importantly, it is found that for the NDs-Cu2+ exposure groups, large amount of Cu2+ inside the cells were mainly attached to NDs, illustrating that Cu2+ entered the cells in the form of ND-Cu2+ complexes. Another synchrotron-based μXRF experiment was performed to further examine the difference in intracellular Cu2+ concentration with or without NDs. Fluorescence spectra showed that cells cultured in medium containing 25 μg/mL Cu2+ yielded a ≈ 18 fold increase Cu Kа signal at 8.05 KeV compared with cells cultured in basal medium (see Additional file 1: Figure S8). Consistent with recent literature [34], we found that the total amount of Zn varied little in most of the samples and the Zn concentration followed the cell shape. Additionally, uptake of NDs-Cu2+ complex may influence Zn ion level inside cells due to toxicity. Elemental maps of a typical cell showed that the Cu2+ concentration in cells treated with ND-Cu2+ mixture was significantly higher than that in cells treated with Cu2+ alone, and no Cu2+ signal was detected in cells treated with NDs alone, which was consistent with that treated with basal medium (Figure 3b). Further transmission electron microscopy (TEM) observation as well as energy dispersive spectroscopy (EDS) analysis confirmed these X-ray imaging results (see Additional file 1: Figure S9). Moreover, both STXM and μXRF images showed that addition of NDs made the Cu-rich zone inside cells get more concentrated.Figure 3

Bottom Line: In addition, theoretical calculation and molecular dynamics (MD) computation were used to illustrate the adsorption properties of different metal ion on NDs as well as release profile of ion from ND-ion complexes at different pH values.Detailed investigation of ND-Cu2+ interaction showed that the amount of released Cu2+ from ND-Cu2+ complexes at acidic lysosomal conditions was much higher than that at neutral conditions, leading to the elevation of intracellular ROS level, which triggered cytotoxicity.The present experimental and theoretical results provide useful insight into understanding of cytotoxicity triggered by nanoparticle-ion interactions, and open new ways in the interpretation of nanotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Division of Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201800, China. zhuying@sinap.ac.cn.

ABSTRACT

Background: Nanomaterials hold great promise for applications in the delivery of various molecules with poor cell penetration, yet its potential for delivery of metal ions is rarely considered. Particularly, there is limited insight about the cytotoxicity triggered by nanoparticle-ion interactions. Oxidative stress is one of the major toxicological mechanisms for nanomaterials, and we propose that it may also contribute to nanoparticle-ion complexes induced cytotoxicity.

Methods: To explore the potential of nanodiamonds (NDs) as vehicles for metal ion delivery, we used a broad range of experimental techniques that aimed at getting a comprehensive assessment of cell responses after exposure of NDs, metal ions, or ND-ion mixture: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Trypan blue exclusion text, optical microscope observation, synchrotron-based scanning transmission X-ray microscopy (STXM) and micro X-ray fluorescence (μXRF) microscopy, inductively coupled plasma-mass spectrometry (ICP-MS), reactive oxygen species (ROS) assay and transmission electron microscopy (TEM) observation. In addition, theoretical calculation and molecular dynamics (MD) computation were used to illustrate the adsorption properties of different metal ion on NDs as well as release profile of ion from ND-ion complexes at different pH values.

Results: The adsorption capacity of NDs for different metal ions was different, and the adsorption for Cu2+ was the most strong among divalent metal ions. These different ND-ion complexes then had different cytotoxicity by influencing the subsequent cellular responses. Detailed investigation of ND-Cu2+ interaction showed that the amount of released Cu2+ from ND-Cu2+ complexes at acidic lysosomal conditions was much higher than that at neutral conditions, leading to the elevation of intracellular ROS level, which triggered cytotoxicity. By theoretical approaches, we demonstrated that the functional carbon surface and cluster structures of NDs made them good vehicles for metal ions delivery.

Conclusions: NDs played the Trojan horse role by allowing large amounts of metal ions accumulate into living cells followed by subsequent release of ions in the interior of cells, which then led to cytotoxicity. The present experimental and theoretical results provide useful insight into understanding of cytotoxicity triggered by nanoparticle-ion interactions, and open new ways in the interpretation of nanotoxicity.

No MeSH data available.


Related in: MedlinePlus