Quantum Dots Elicit Hepatotoxicity through Lysosome-Dependent

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Quantum Dots elicited Hepatotoxicity through Lysosomedependent Autophagy Activation and ROS Production Jiajun Fan, Shaofei Wang, Xuyao Zhang, Wei Chen, Yubin Li, Ping Yang, Zhonglian Cao, Yichen Wang, Weiyue Lu, and Dianwen Ju ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00824 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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ACS Biomaterials Science & Engineering

Quantum Dots elicited Hepatotoxicity through Lysosome-dependent Autophagy Activation and ROS Production Jiajun Fan1, 2#, Shaofei Wang1, 2#, Xuyao Zhang1, 2#, Wei Chen1, 2, Yubin Li1, 2, Ping Yang3, Zhonglian Cao3, Yichen Wang1, 2, Weiyue Lu2, Dianwen Ju1, 2*

1.

Minhang

Branch,

Zhongshan

Hospital,

Fudan

University/Institute

of

Fudan-Minhang Academic Health System, Minhang Hosptial, Fudan Univeristy 2. Department of Microbiological and Biochemical Pharmacy & Key Lab of Smart Drug Delivery MOE, School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China 3. Instrumental Analysis Center, School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China

# Jiajun Fan, Shaofei Wang and Xuyao Zhang contributed equally to this work Corresponding Author * Dianwen Ju, Ph.D.

E-mail: [email protected]; Tel: +86 21 51980037; Fax:

+86 21 51980036.

ACS Paragon Plus Environment

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Abstract Quantum dots were reported to be metabolized by liver and demonstrated to be toxic in vitro and in vivo with unclear mechanisms, which largely limited its applications in the field of biomedical research. To improve its biosafety, the mechanism of how the quantum dots triggered hepatotoxicity was evaluated in this study. We found that CdTe/CdS quantum dots (QDs) could trigger significant apoptosis-independent nanotoxicity after its uptake by liver cells and internalization into lysosomes. Besides, the lysosomal enzymes were abnormally activated after the QDs entered the lysosomes, which caused ROS production and autophagy activation. Importantly, inhibition of lysosomal enzymes not only rescued the viability of liver cells but also blocked the production of ROS and activation of autophagic flux, whereas inhibition of ROS and autophagy could ameliorate the hepatotoxicity induced by QDs but had no impact on the activity of lysosomal enzyme. Our results elucidated the relationship among the lysosomes, ROS and autophagy in QDs-induced hepatotoxicity, which indicated that the QDs could elicit hepatotoxicity through lysosome-dependent autophagy activation and ROS production, highlighting an approach to improve the biosafety of quantum dots by lysosomal inhibition.

Keywords: Quantum Dots, ROS, Autophagy, Lysosome, Nanotoxicity


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Introduction Liver, being the target of nanomaterials (NMs) and an important place for NM metabolism, appeared to have a higher frequency than other tissues to interact with NMs1, 2. In the previous studies, a series of nanomaterials including nano-copper particles 3, ZnO particles

4, 5

and silver nanomaterials

6, 7

were demonstrated to elicit

remarkable hepatotoxicity in vitro and in vivo. Unfortunately, the quantum dots have no exceptions 8. Several types of quantum dots like graphene quantum dots Cd/Se/Te-based ones

11, 12

9, 10

and

have been demonstrated to be severely toxic to liver. They

could induce significant apoptosis or necrosis of liver cells, and modulate redox homeostasis in liver tissues 13-16. Although the quantum dots had shown their values in the application of diagnosis and live-animal imaging, considering their potential hazards to the liver, the applications of the quantum dots for biomedical and clinical use were restricted

17, 18

. To reduce the toxicity of quantum dots, their biological

effects, including their cellular uptake

19, 20

and intracellular trafficking

21 ,22

, as well

as the cell death and cell cycle arrest, have been studied in liver cells. It was reported that the quantum dots could promote apoptosis, autophagy and the formation of reactive oxygen species (ROS) in liver cells, resulting in hepatotoxicity 23-25; however, the exact subcellular mechanisms of liver injury induced by these nanocrystals were still unclear. Lysosome, a kind of spherical vesicles containing various enzymes which are able to break down almost all kinds of biomolecules, including proteins, nucleic acids, lipids, carbohydrates and cellular debris, acted as the waste disposal system of eukaryotes through degradation of unwanted molecules in cytoplasm 26-28. Besides its



major functions on cell microenvironment maintaining, lysosomes were also the key organelle that caused cell death 29, 30. Cellular stress such as hypoxia, lack of nutrients and cytotoxic agents could directly result in dysfunction of lysosomes and disrupt the phospholipid bilayers, which caused increased ROS, DNA fragmentation and apoptosis

31-34

. Furthermore, because the majority of NMs entered cells through

endocytosis, lysosomes were also frequently a target for their toxicity 35-37. It was well documented that lysosomes dominated the toxicity of silver nanomaterials on human HepG2 cells

38

and contributed to the inflammatory injury induced by multiwall

carbon nanotubes

39, 40

. Therefore, these evidences indicated that lysosomes were

important for the NM-based nanotoxicity, while the role of lysosomes in quantum dots-induced hepatotoxicity was still unclear. Thus, further evaluation against the relationship between the lysosomes and hepatotoxicity was required to offer a further understanding on the subcellular mechanism of quantum dots-induced hepatotoxicity. Quantum Dots CdTe/CdS 569 (QDs), a typical cadmium-based quantum dots, were generated and used to investigate the role of lysosomes in quantum dots-induced hepatotoxicity. In this study, we investigated at the cellular and subcellular level the interaction between QDs and liver cells, including its uptake by cells and internalization by lysosomes, as well as the cytotoxic effects like ROS production and autophagy initiation. Importantly, we revealed the linkage between the abnormal activation of lysosome function and hepatotoxicity of QDs. We finally evaluated the relationship among the lysosomes, ROS and autophagy, which demonstrated that the QDs elicit nanotoxicity mainly through lysosome-based ROS production and


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autophagy activation and highlighted an approach to decrease the hepatotoxicity of quantum dots by lysosomal inhibition.

Materials and Methods Materials Cyto-ID green dye autophagy detection kit was purchased from Enzo Life Sciences, Inc (Farmingdale, NY, USA), MitoSox ROS detection kit was obtained from Invitrogen™ (Grand Island, NY). All other reagents were purchased from Sigma (St Louis, MO, USA).

Synthesis of the QDs The QDs was prepared according to the previous research 41. Briefly, CdTe cores were established in aqueous solution (pH 9.0) by CdCl2, fresh NaHTe and MPA as precursors. All the reactions were maintained at 95 °C for 12 h. To coat with a CdS shell, Na2S solutions were then added into the CdTe solutions and maintained at 60 °C for 1.5 h, and then stored at 2 °C. The molar ratio of Cd:Te:S:MPA in the reaction solution was 1:0.4:0.012:4.16. All the reactions were performed under the protection of N2 atmosphere. The MPA-coated CdTe/CdS core/shell 569 with maximum emissions at 569 nm, were employed for further research on its hepatotoxicity.

Characterization The fluorescence and emission spectra were determined by using a fluorescence



spectrophotometer equipped with a xenon lamp (F-2500; Hitachi, Tokyo, Japan). Excitation spectra of QDs in different solutions were tested from a wavelength region of 200–800 nm, and the emission spectra were collected from a wavelength region of 400–800 nm. Particle size and zeta potential of the QDs were measured by photon correlation spectroscopy (PCS) using Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK). The size distribution data were analyzed with respect to their intensity.

Cell Culture Human normal liver cell line HL-7702 cells and human liver hepatocellular carcinoma cell line HepG2 cells were obtained from Cell Bank of Chinese Academy of Sciences, Shanghai Branch (Shanghai, China), and routinely cultured in RPMI1640 and DMEM medium, respectively, supplemented with 10% of fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Cells were maintained at 37°C in environment of 95% air and 5% CO2. For experimental use, QDs and inhibitors of lysosome/autophagy/ROS were prepared and diluted by corresponding medium.

Confocal Microscopy Cells were seeded at approximately 10,000 cells/well in confocal plates (NEST Biotechnology Co., Ltd., Jiangsu, China) and pre-treated as was described 41-43. Then samples were stained for immunofluorescence confocal assay using Cyto-ID® Autophagy Detection Kit and/or MitoSox as recommended by the manufacturer.


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Merged images were obtained according to the Recommended Assay Procedure using AttovisionTM software (Becton, Dickinson and Company, New Jersey, USA).

Cellular Lysosomal Enzyme Activity Assays Cellular lysosomal enzyme activity was measured accordingly. Liver cells were treated with or without QDs for 12 h. Afterwards, cells were solubilized by adding 25 µL of 0.1% Triton X-100 and incubated for 30 min at room temperature. Then, 150 µL of 10mm p-nitro-phenyl-phosphate was added to each well as a substrate for acid phosphatase, followed by the addition of 50µL of citrate buffer. After incubation for 1 h at 37 oC, 50 µL of borate buffer was added to the mixture to stop the reaction. Optical densities were measured at 405 nm to detect the relative lysosomal enzyme activity.

MTT-based Cell Viability Assays Cell viability was determined by using MTT-based assay. Cells in exponential growth phase were seeded into 96-well plates at the concentration of 10000-20000 cells/mL. QDs (or Cd2+) with or without lysosome/autophagy/ROS inhibitors were thus added to cultures. Cells treated with and the complete culture medium was used as a control. Then 10µL of MTT solution were mixed to each culture after co-incubation with QDs and autophagy inhibitors. Cells were maintained at 37°C in environment of 5% CO2 for 4h and the supernatant was abandoned. Afterwards, 100µL of dimethyl sulfoxide were then added to dissolve formazan. The optical



density (O.D.) values at wavelength 570 nm were determined to calculate the relative number of surviving cells. The results were normalized to that of negative control and expressed as a percentage of the O.D. value of the control.

Flow Cytometry (FCM) Assay After cells were incubated with or without different concentrations QDs for 24h, cellular uptake of NMs was measured by FCM assays.

Statistics Statistics analysis was carried out with GraphPad Prism 5. The results were expressed as means ± SD. All the experiments have been done at least 3 times and comparisons were performed using Student’s t test (two-tailed). P-value 0.05



Figure 1 173x189mm (300 x 300 DPI)


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Figure 2 173x220mm (300 x 300 DPI)



Figure 3 173x147mm (300 x 300 DPI)


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Figure 4 189x139mm (300 x 300 DPI)



Figure 5 173x162mm (300 x 300 DPI)


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Figure 6 173x117mm (300 x 300 DPI)



Table of Contents Graphic 66x60mm (300 x 300 DPI)


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Quantum Dots Elicit Hepatotoxicity through Lysosome-Dependent

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