Supplementary Information

Solubility of nano-sized metal oxides evaluated by using in vitro simulated

lung and gastrointestinal fluids: Implication for health risks

Laijin Zhong, Yanlin Yu, Hong-zhen Lian*, Xin Hu*, Haomin Fu, Yi-jun Chen

State Key Laboratory of Analytical Chemistry for Life Science, Collaborative Innovation

Center of Chemistry for Life Sciences, School of Chemistry & Chemical Engineering and

Centre of Materials Analysis, Nanjing University, Nanjing 210023, P.R. China

Table S1. Information of the commercial nanoparticles used in this work referring to

http://www.sigmaaldrich.com; http://www.reagent.com.cn

Product name Manufacturer

Product/

Lot numberForm Purity

(%)Size labelled

(nm)Size measured by XRD (nm)*

nZnO Sigma-aldrich677450/

MKBX1354VNanopowder >97 <50 (BET) 25.83

nCuO Sigma-aldrich544868/

MKBT8894VNanopowder 96.6 <50 (TEM) 17.18

nCeO2 Sigma-aldrich544841/

MKBK4359VNanopowder Not

marked <25 (BET) 28.97

nTiO2 Sigma-aldrich634662/

MKBG7739VNanopowder 99.5 <50 (XRD) 25.16

nFe3O4Sinopharm

Reagent I109514 Nanopowder 99.5 20 (BET) 41.24

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* The data were measured in this study.

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Table S2. Chemical composition of artificial lysosomal fluid (ALF, pH 4.5) (Colombo, et al.

2008)

Chemical constituents Amount (g L-1)

Magnesium chloride (MgCl2) 0.050

Sodium chloride (NaCl) 3.210

Calcium Chloride (CaCl2) 0.128

Sodium sulphate (Na2SO4) 0.039

Disodium hydrogen phosphate (Na2HPO4) 0.071

Sodium citrate dihydrate (NaH2C6H5O7·2H2O) 0.077

Sodium hydroxide (NaOH) 6.000

Citric acid (C6H8O7) 20.800

Glycine (NH2CH2COOH) 0.059

Sodium tartrate dihydrate (C4H4O6Na2·2H2O) 0.090

Sodium lactate (C3H5NaO3) 0.085

Sodium pyruvate (C3H3O3Na) 0.086

Table S3. Chemical composition of Gamble solution (pH 7.3) (Juhasz, et al. 2009, Wragg, et

al. 2007)

Chemical constituents Amount (g L-1)

NH4Cl 0.535

NaCl 6.786

CaCl2 0.022

H2SO4 0.045

NaH2PO4 0.144

NaHCO3 2.268

Sodium citrate (CH3CHOHCOONa) 0.052

Glycine (NH2CH2COOH) 0.375

L-Cysteine (C3H7NO2S) 0.121

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Pentetic acid (DTPA, C14H23N3O10) 0.079

Benzalkonium chloride (ABDAC, C21H38NCl) 0.050

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Table S4. Chemical composition of solubility bioavailability research consortium (SBRC)

and in-vitro gastrointestinal (IVG) method (Schroder, et al. 2004)

SBRC IVG

Gastric s olution

pH 1.5 1.8

Glycine (NH2CH2COOH) (g L-1) 30.00 None

NaCl (g L-1) None 8.77

Pepsin (g L-1) None 10.00

Intestinal s olution

pH 7.0 5.5

Pancreatin (g L-1) 0.50 0.35

Bile extract (g L-1) 1.75 3.50

Formula S1. Stokes equation (Katkov, et al. 1999):

t=¿¿

Table S5. The lists of parameters in Stokes equation

Parameters Contents Unit

t The centrifugation time s

η The viscosity of solvent g cm-1 s-1

R1 The distance between the axis of a rotor to the liquid level centrifuging cm

R2 The distance between the axis of a rotor to the precipitating dots cm

N The rotate speed rps

r The radius of nanoparticles cm

dp The density of particle g cm-3

d The density of solvent g cm-3

θ The inclination angle of the tube in a rotor °

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To ensure these nano-sized particles precipitated thoroughly at 10000 rpm for 10 min,

the Stokes equation was a trial. Centrifugation time of these nano-sized particles was

calculated under 10000 rpm with the high speed centrifuge Model CT14D Tabletop

(Techcomp Shanghai Co., Ltd.). The results are shown as follows.

The parameters and centrifugation time

The CT14D Rotor (TA15-8-10) Structure Chart

Parameters η (g cm-1 s-1) d (g cm-3) R1 (cm) R2 (cm) N (r s-1) θ (°)

Value0.01005

(20 oC, Water)

0.99823

(Water)6.0 8.0 166.7 30

Nanoparticles ZnO CuO TiO2 CeO2 Fe3O4

dp (g cm-3) 5.6 6.3 3.9 7.1 5.2

t’ (min) 5.9 11.7 7.4 3.6 2.6

Note: The radius of nanoparticles r is referred to Figure S1.

According to the Stokes equation, 10 min is more enough to most of these nanoparticles

to separate from water. Despite of the higher viscosity and the different density of simulated

biological fluids, the centrifugation time is much less than the theory value considering to the

aggregation effect which is a well-known phenomenon in nano-sized particles’ suspension. In

fact, 5 ml 10 mg L-1 nCuO suspension is separated entirely under 10000 rpm for 5 min with

the weak Cu2+ signal of the acidified supernatant measured by ICP-OES.

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Table S6. The instrumental parameters of inductively coupled plasma optical emission

spectrometer (ICP-OES, Perkin-Elmer SCIEX, Optima 5300)

Conditions Parameters

Plasma argon flow 15 L/min

Auxiliary argon flow 0.2 L/min

Nebulizer argon flow 0.8 L/min

Sample flow rate 1.50 L/min

Spray chamber Cyclonic spray chamber

Nebulizer Concentric glass nebulizer

Viewing position (15, 0)

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Formula S2. Bioaccessibility (Huang, et al. 2016):

Bioaccessibility , % =CM·V·F m·W·1000

×100

Where CM is the average concentration of metal measurement by ICP-OES, mg L -1; V is

the dosage of the biofluid, 5 ml; F is the dilute factor, m is the dose of particulates, 50 mg; W

is the mass percentage of the metal in the compound.

Table S7. The bioaccessibility (%) of metals for nano-sized metal oxides (Mean±SD).

Method nZnO nCuO nCeO2 nFe3O4 nTiO2

Gastric SBRC 79.9±4.6 24.4±0.6 5.5±0.1 2.0E-03±0.2E-03 7.5E-03±0.7E-03

Intestinal SBRC 15.4±0.9 13.8±0.2 N/A 1.3E-03±0.1E-03 0.9±0.0

Gastric IVG 9.1±0.3 1.1±0.1 0.2±0.0 3.1E-02±0.1E-02 5.8E-05±0.6E-06

Intestinal IVG 2.6±0.1 0.2±0.0 0.2±0.0 3.0E-03±1.3E-03 9.6E-06±1.4E-06

ALF 4.0±0.3 0.4±0.0 5.2E-03±0.0 2.1E-03±0.1E-03 1.2E-03±0.1E-03

Gamble solution 0.5±0.0 1.6±0.1 7.4E-02±0.4E-02 1.1E-05±0.3E-05 7.3E-03±0.9E-03

Note: Values are measured at 2 h for gastric phase, 4 h for intestinal phase and 24 h for ALF and Gamble

solution (n=3).

Table S8. The bioaccessible concentrations (mg L-1) of metals in the simulated lung and

gastrointestinal fluids

Methods nZnO nCuO nCeO2 nFe3O4 nTiO2 ZnO ZnSiO3 ZnS CuO

Gastric SBRC 6410 1947 446.4 0.1760 0.4427 6278 2989 0.4237 1247

Gastric IVG 726.1 89.87 17.21 2.187 0.02133 254.3 157.7 1.128 117.3

Intestinal SBRC 6152 1099 N/A 0.1013 56.25 8713 4069 0.3093 368.5

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Intestinal IVG 225.1 18.84 18.70 0.2133 0.2200 573.0 54.57 0.4080 25.20

ALF 322.6 30.12 0.400 0.1333 0.07733 401.8 9.515 0.730

7 22.34

Gamble solution 38.78 124.3 6.024 0.1013 0.01600 17.07 6.987 2.011 50.83

Note: Values are measured at 4 h for intestinal phase and 24 h for ALF and Gamble solution (n=3). The

digits in bold represent maximal values, and the digits in italic represent minimal values.

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Figure S1. XRD patterns of the five nano-sized metal oxides and their size calculated by

using Scherrer formula (Rao, et al. 2008): D=K γ β-1 cosθ-1. D is the average diameter of the

measured particle (nm); K is the Scherrer factor (K=0.89 in this case); γ is wavelength of the

X-ray (0.154056 nm); β is the peak width at half height (rad); θ is the angle of diffraction

(rad).

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Figure S2. The hydrodynamic radius of the nano-sized metal oxides in simulated

physiological fluids monitoring by using dynamic light scattering (the hydrodynamic radius

of nZnO in Gastric SBRC only obtained at the initial state).

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Figure S3. The ratios of final to initial absorbance of the nano-sized metal oxides suspended

in the simulated biological fluids monitoring by using UV-Vis spectrometry.

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Figure S4. Zeta potential of nano-sized metal oxides. The values of Zeta potential are ranged

from -20 mV to 10 mV.

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Figure S5. The kinetic effect of bioaccessibility.

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