Determining the Cytotoxicity of Rare Earth Element Nanoparticles in

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Determining the toxicity of rare earth oxide nanomaterials in macrophages and the involvement of membrane damage Jie Gao, Ruibin Li, Fengbang Wang, Xiaolei Liu, Jie Zhang, Ligang Hu, Jianbo Shi, Bin He, Qunfang Zhou, Maoyong Song, Bin Zhang, Guangbo Qu, Sijin Liu, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04231 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 12, 2017

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Environmental Science & Technology

Sequestration of membrane phosphates by toxic REE NPs

Cell membrane disruption

Elevated intracellular

Ca2+

Elevated mitochondrial Ca2+

Lysosomal membrane disruption

Released lysosomal enzyme into cytosol

Cell necrosis ACS Paragon Plus Environment


1

Determining the cytotoxicity of rare earth element nanoparticles in macrophages

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and the involvement of membrane damage

3

Jie Gao†,‡, Ruibin Li§, Fengbang Wang†,‡, Xiaolei Liu†,‡, Jie Zhang†,‡, Ligang Hu†,‡,

4

Jianbo Shi†,‡, Bin He†,‡, Qunfang Zhou†,‡, Maoyong Song†,‡, Bin Zhang#, Guangbo

5

Qu*,†,‡, Sijin Liu†,‡, Guibin Jiang†,‡

6

†

7

Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

8

100085, P. R. China

9

§

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative

10

Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions,

11

Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, Soochow

12

University, Suzhou, Jiangsu 215123, P. R. China

13

#

14

250100, China

15

‡

School of Chemistry and Chemical Engineering, Shandong University, Jinan

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

16 17 18 19 20 21 22 23 24 25

Corresponding author:

26

Guangbo Qu, Ph.D, Email: [email protected]

27

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ACS Paragon Plus Environment

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Abstract

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Rare earth oxide nanomaterials (REE NPs) hold considerable promise, with high

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availability and potential applications as superconductors, imaging agents, glass

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additives, fertilizers additives and feed additives. These results in potential REE NP

32

exposure to humans and the environment through different routes and adverse effects

33

induced by biological application of these materials are becoming an increasing

34

concern. This study investigates the cytotoxicity of REE NPs: nLa2O3, nEu2O3,

35

nDy2O3 and nYb2O3 from 2.5 µg/mL to 80 µg/mL, in macrophages. A significant

36

difference was observed in the extent of cytotoxicity induced in macrophages by

37

differential REE NPs. The high-atomic number materials (i.e. nYb2O3) tending to be

38

no toxic while low-atomic number materials (nLa2O3 and nEu2O3 and nDy2O3)

39

induces 75.1%, 53.6% and 20.7% dead cells. With nLa2O3 as the representative

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material, we demonstrated that nLa2O3 induced cellular membrane permeabilization,

41

through the sequestration of phosphates from membrane. The further mechanistic

42

investigation established that membrane damage induced intracellular calcium

43

increased to 3.0 to 7.3 fold compared to control cells. This caused the sustained

44

overload of mitochondrial calcium by approximately 2.4 fold, which regulated cell

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necrosis. In addition, the injury of cellular membrane led to the release of cathepsins

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into cytosol which also contributed to cell death. This detailed investigation of

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signaling pathways driving REE NP-induced toxicity to macrophages is essential for

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better understanding of their potential health risks to humans and the environment.

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Introduction

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Owing to their unique physical-chemical properties, rare earth elements (REEs)

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have been extensively applied as superconductors, imaging agents, glass additives,

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fertilizers additives and feed additives, etc.1-3 For instance, addition of Eu2O3 to ZnO

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films could improve the optical and structural properties and intense luminescence.4

55

Yb2O3 has been reported as a novel long circulation material as an in vivo CT

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imaging agent.5 Yb2O3 could be applied for novel additive material for concentrator

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solar cells, as a the solar energy converter.6 Dy2O3 nanoparticles mixed

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gadolinium-dysprosium oxide nanoparticles showed promising characteristics as a

59

MRI contrast agent for imaging.7 It has been reported that fertilizers contain

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approximate 30% of La2O3. Commercial feed additives for poultry containing 50 %

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REEs oxides earth and La2O3 accounts for 20% of REE oxides.8

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With the increase in application of REEs worldwide, thousands of tons of REEs

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enriched fertilizers released into the agricultural soil, resulting in the large scale of

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contamination of REEs in soil and water near the mining sites REEs.3,

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instance, because of the large REE reserves, China is the largest worldwide producer

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and supplier of REEs.9 It has been evaluated in 2002 that REE containing fertilizers

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discharged into the cultivated soil are above 5000 tons in China.3 Therefore,

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application of REEs has enhanced the large scale contamination of REEs, displaying

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high accumulative in contaminated environmental compartments, and been considered

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persistence in environment and bioaccumulation in biota.15 REEs containing food

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products, industrial products or medicinal agents could be intake through oral,

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intravenous injection and skin exposure.16 The exposed REEs could accumulated in

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blood, brain and bone after introducing to human body,17 leading to a great concern for

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human health.18-21 The inhalation of air-borne particles of REEs oxides is the most

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typical accidental exposure route.22 Because of the chronic ingestion of REEs, the

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population living in high-REE-background showed abnormality in activities of some Page 3 of 34


10-14

For

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digestive enzymes.23 As various nanomaterials containing REEs have been developed

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as probes for imaging,24-28 these direct administrations of these REEs containing

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nanomaterials, therefore, may also result in a potential risk to human health. It has

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been reported that fibrosis and renal disorders in patients when Gd elements (9-25

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mmol) used as an imaging agent.18, 29

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NPs) present remarkably different biological activities in comparison to their

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larger-scale counterparts, with the higher surface area significantly altering cellular

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uptake and bio-distribution in vivo, resulting in altered biological effects.31 Therefore,

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improved understanding of the impacts on biological systems induced by these

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materials is highly beneficial for the prevention of side effects in biomedical

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applications.32

30, 31

At the nanoscale, REE nanoparticles (REE

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Macrophages are considered a vital sentinel cell, forming a defensive line to

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combat invading pathogens and they also take a prominent role in the clearance of

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nanomaterials from circulatory systems and local organs.26 Because of the instinct

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role of macrophage, the high accumulation level of environmental particles in these

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cells and the resulted negative effects are considered the dominant risk upon particle

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exposure. Disordered macrophages not only lose their defense activity, but also have

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been found to trigger various pathologies such as autoimmune diseases,33

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inflammation and pneumonitis.34 For instance, atmospheric particles could be cleared

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by macrophages located in lung and the accumulation of particle in macrophages

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leads to sustainable inflammation, and the resulted in lung diseases.35,

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environmental exposure of atmospheric particle has been demonstrated to trigger lung

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cancer due to the inflammation initiated by lung macrophages.35, 36 Exposure to REE

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NPs has been associated with nephrogenic systemic fibrosis in patients,18 because

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macrophage internalized REE NPs such as La2O3 and Eu2O3 cause the transfer of

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phosphates from lipids, leading to membrane damage and injury to lysosomal cell

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membranes,

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inflammasome.37-43 However, as a lack of complete understanding of the mechanisms

promoting

activation

of

the

multiprotein

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oligomer

36

The

NLRP3


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underlying REE NP effect on macrophages, the impact of REE NPs on cell

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survival and viability is at present still unclear. Therefore, the investigation on the

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toxicity of REE NPs with macrophage as cell model is of help to evaluate their risks

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under the exposure REE NPs by various routes.

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Physicochemical properties such as elemental composition, shape, size and

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surface groups largely dictate the toxicity profiles of nanomaterials.26 Therefore,

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different REE NPs are therefore assumed to induce distinct biological effects. In the

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present study, we investigated the cytotoxicity of REE NPs on the survival of

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macrophages, establishing potential mechanisms responsible for the cytotoxicity

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induced by these materials. These findings show a safer pathway for the design of

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RE-containing nanomaterials, for application in biomedical systems.

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Materials and Methods

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Chemicals and reagents

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Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin and

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L-glutamine, were all purchased from Invitrogen (Carlsbad, USA). Fetal bovine

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serum (FBS) was obtained from Atlanta Biologicals Inc. (Lawrenceville, USA). All

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horseradish peroxidase-conjugated secondary antibodies were obtained from GE

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Healthcare Life Sciences (Pittsburgh, USA). The mammalian protein extraction

123

reagent and ECL reagent were purchased from Thermo Fisher Scientific (Waltham,

124

MA, USA). The protein inhibitor cocktail, 2',7'-Dichlorodihydrofluorescein diacetate

125

(DCFH-DA), rapamycin, and tetramethylrhodamine methyl ester (TMRM) were

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purchased from Sigma Aldrich (Shanghai, China). The Bradford reagents for protein

127

determination were obtained from Bio-Rad (Shanghai, China). The following

128

antibodies (Abs) were used: anti-Bid Ab from R&D Systems (Minneapolis, USA),

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anti-LC-3 Ab from Novagen (Medison, USA), while anti-GAPDH and anti-Caspase 3

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Ab were purchased from Cell Signaling Technology (Shanghai, China). Page 5 of 34


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4-(6-Acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy)

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dianiline-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester (Fluo-3/AM),

133

9-[4-[Bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-3-[2-[2-[bis[2-[(acetyloxy)meth

134

oxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-3,6-bis(dimethylamino)xa

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nthylium bromide(Rhod-2), E-64d, Pepstatin A, CA-074 methyl ester (CA-074Me),

136

Cyclosporin A (CsA), Cytochalasin D, ethylenediaminetetraacetic acid (EDTA) and

137

Z-VAD-fmk were purchased from MedChem Express (Monmouth Junction, USA).

138

Nanomaterials and characterization

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All REE NPs including La2O3 NP (nLa2O3), nEu2O3, nDy2O3, and nYb2O3

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(sources of REE NPs was shown in Table S1) were prepared in endotoxin-free water

141

forming stock solutions of 5 mg/mL. Before characterization or exposure, REE NPs

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were sonicated at 32 W for 30 s. High-resolution transmission electron microscopy

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(HRTEM) samples were prepared by depositing of REE NP suspension (20 µg/mL)

144

onto a carbon-coated copper electron microscopy grid (Beijing Zhongjingkeyi

145

Technology Co., Ltd., China) and dried under ambient conditions overnight before

146

analysis using a HRTEM (JEOL-2100F, Japan). Zeta potential and hydrodynamic

147

diameters of REE NPs in DMEM medium were determined using a Zetasizer (Malvern

148

Nano series, Malvern, U.K.). Fourier transform infrared spectroscopy (FT-IR) was

149

performed using a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher Scientific

150

Inc., USA), according to the KBr pellet method. X-ray photoelectron spectroscopy

151

(XPS) determination of REE NPs was performed using sample deposits on silicon

152

wafers, where samples were air-dried prior to analysis.

153

Cell Culture, cellular treatment and toxicity assays

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J774A.1 cells and Hep1-6 cells were purchased from the Shanghai Cell Bank of

155

Type Culture Collection of China. Following treatment at 2.5, 10, 20, 40, and 80

156

µg/mL,

157

phosphate-buffered saline (PBS), with dead cells analyzed by flow cytometric

cells

were

stained

using

50

µg/mL

propidium

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iodide

(PI)

in


158

analysis.38,

44-46

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cytotoxicity according to manufacturer instructions (Promega, Shanghai, China).

The CellTox™ Green Cytotoxicity assay was used to determine

160

J774A.1 cells were pre-incubated with 1 µM cytochalasin D, 50 µM EDTA or 2

161

nM rapamycin for 2 h, respectively, followed by exposure to 20 µg/mL nLa2O3 with

162

or without cytochalasin D, EDTA or rapamycin. Cell death was then evaluated using

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PI staining after 24 h.

164

RAW-DifluoTM mLC3 cells (invivogen) were cultured in DMEM containing 10%

165

(v/v) FBS, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml NormocinTM, and 2

166

mM L-glutamine. Before the exposure experiment, the culture medium was changed

167

with DMEM containing heat-inactivated FBS (30 min at 56 ℃), 50 U/ml penicillin,

168

50 mg/ml streptomycin, 2 mM L-glutamine, and 4.5 g/L glucose, 10% (v/v). After 24

169

h, RAW-DifluoTM mLC3 cells was treated with nLa2O3 for 6 h followed by the

170

observation with confocal microscopy.

171

Inductively coupled plasma-mass spectrometry (ICP-MS) analysis for cellular

172

uptake of nanomaterials

173

Cells were cultured in 10-cm plates and treated with 20 µg/mL REE NPs for 6 h.

174

Cells were washed 3 times with PBS. Then we collected and digested the cells by

175

combining with RIPA buffer containing 1% protease cocktail (Sigma Aldrich) on ice

176

for 30 min. After centrifugation at 15,000 g for 30 min at 4 ºC, the protein

177

concentrations of the supernatant of cellular lysates were determined using the

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Bradford method. The suspension of cell lysates post centrifugation ICP-MS or the

179

pellets of cellular lysates were digested for ICP-MS determination. In brief, 200 µL of

180

supernatants or pellets were digested with 200 µL of nitric acid-H2O2 (3:1) at 75 ºC

181

for 12 h, respectively. The digested solutions were diluted to 10 mL with the acid

182

concentration below 3% (v/v) and subjected to ICP-MS analysis to determine the

183

quantity of particulate REEs (La, Eu, Dy, and Yb). All analyses were performed in

184

triplicates using the external calibration method. The standard mixture solution for Page 7 of 34


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REE determination was purchased from Guobiao (Beijing) Testing & Certification

186

Co., Lta. (Beijing, China). The calibration curve was shown in Figure S1 (Supporting

187

Information). Linear regressions showed that R was above 0.99 (Figure S1). The

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signal intensity repeatability (RSD) of standard was 9.4%. Each group include at least

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3 parallel samples and the average value was calculated. The sum of the dissolved

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REE and particulate REE content is defined as the total intracellular REE

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concentration. The intracellular total REEs concentration was expressed as REE

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content (ng) versus intracellular protein level (mg).

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Transformation of REE NPs in phagolysosomal simulant fluid (PSF)

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Nanoparticles were dissolved in 1 mL PSF solution (142 mg/L Na2HPO4, 6.65

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g/L NaCl, 62 mg/L Na2SO4, 29 mg/L CaCl2∙H2O, 250 mg/L glycine, 8.09 g/L

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potassium phthalate, pH 4.5) at a concentration of 50 µg/mL and sonicated at 32 W

197

for 30 s. Samples were incubated at 37 °C for either 0.25, 0.5, 1, 3, or 6 h, prior to

198

centrifugation at 15,000 g for 30 min. Pellets were analyzed by inductively coupled

199

plasma-optical emission spectrometry (ICP-OES) to assess the concentrations of

200

phosphorous and REE ions, while suspensions were digested and REE ion

201

concentrations were determined by ICP-MS.38

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Assessment of lysosomal membrane permeabilization (LMP)

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Regarding concentration selection, for the investigation of the cytotoxicity of

204

nLa2O3 as the representative material, 20 µg/mL was frequently selected because the

205

concentration higher than 20 µg/mL (40 or 80 µg/mL) possess dramatic toxicity and

206

could induced cell detached from the tissue culture dish. As a result, some experiments

207

such as confocal fluorescence observation could not performed under the exposure

208

concentration higher than 20 µg/mL. After exposure to 20 µg/mL nLa2O3 in DMEM

209

for 6 h, J774A.1 cells were washed with PBS and then stained with Magic Red™

210

Cathepsin B (ImmunoChemistry Technologies, Bloomington, USA) for 2 h according

211

to manufacturers’ instructions. The plasma membrane was stained using a membrane Page 8 of 34



212

labeling kit (Sigma Aldrich, Shanghai, China) and nuclei were stained with Hoechst

213

33342 (Thermo Fisher Scientific, Waltham, MA, USA), at room temperature for 1 h.

214

Finally, cells were visualized using a TCS SP5 laser scanning confocal microscope

215

(Leica, Germany).

216

Calcium flux analysis and mitochondrial potential assessment

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Alterations to concentrations of intracellular Ca2+ and mitochondrial Ca2+ were

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monitored using Fluo-3/AM and Rhod-2 fluorescence probes, respectively. Briefly,

219

after treatment nLa2O3 at 20 µg/mL 1 × 105 cells were incubated at 37 °C in the dark

220

with 4 µM Fluo-3/AM and 4 µM Rhod-2 for 20 min. Cells were then washed 3 times

221

and re-suspended in Hank’s Balanced Salt Solution without Ca2+ and Mg2+. Cells

222

treated with REE NPs were additionally stained with 20 nM TMRM, then analysed by

223

flow cytometry (FACS) for mitochondrial membrane assessment. FACS analysis was

224

performed using an LSR II flow cytometer (Becton Dickinson, Mountain View, CA)

225

equipped with a single 488 nm argon laser. Fluo-3/AM fluorescence was assessed

226

using the fluorescein isothiocyanate (FITC) channel; while PI, TMRM and Rhod-2

227

fluorescence were examined using the PE channel; and both forward and side scatters

228

were used to eliminate cellular fragments. Following treatment, a portion of cells

229

were also fixed with 10% PBS-buffered formaldehyde with 0.1% Triton X-100 for 10

230

min, then stained with 4’ 6-diamidino-2-phenylindole (DAPI) (blue) and Fluo-3/AM

231

(green) or Rhod-2 (red). Fluorescent images were visualized through confocal laser

232

scanning microscopy as described previously.40

233

TEM observation on cell samples

234

After treatment with REE NPs at 20 µg/mL for 6 h, cells were harvested and

235

fixed in 2.5% glutaraldehyde for 2 h, then washed with PBS buffer. Staining was

236

performed using 1% OsO4 for 1 h, prior to dehydration of fixed and stained cells

237

using alcohol (40, 50, 70, 80, 90, 95, or 100% ethanol). Cells were then submerged in Page 9 of 34


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propylene oxide for two 30 min cycled and were then embedded in beem capsules

239

containing pure resin. Blocks were hardened at 70 °C for a 2-day period, then 70 nm

240

sections were cut and stained with 1% lead citrate and 0.5% uranyl acetate, prior to

241

examination using a JEOL JEM 2010F high-resolution transmission electron

242

microscope (Hitachi Scientific Instruments, Japan).

243

Western blot analysis

244

The supernatant of cellular lysates was (20 µg) subjected to both sodium

245

dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting,

246

according to established protocols, as previously described.40 Western blotting signals

247

were detected using a BIO-RAD ChemiDoc XRS chemiluminescence system

248

(Bio-Rad Inc., CA, USA).

249

Statistical Analysis.

250

One-way analysis of variance (ANOVA) was utilized for the analysis of mean

251

differences between two or more treated groups and the untreated control group,

252

while the differences between the two groups were determined using the two-tailed

253

Student's t test. Data are shown in mean ± standard deviation (SD) and pYb, with a similar trend to the ability to for P-REEs

374

complexity. These results indicate the release of dissolved ions from REE NPs is

375

because the interaction between REEs and phosphates. To further evaluate the

376

relation between cell death and dissolved ions levels, the regression analysis was

377

performed. The positive trend was observed between the cell death percentages and

378

the rate of dissolved REEs (Figure S7B, R=0.987, p

Determining the Cytotoxicity of Rare Earth Element Nanoparticles in

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