Impact of in vitro digestion on gastrointestinal fate and uptake of silver nanoparticles with different surface modifications

Figures & data

Table 1. Composition of the artificial digestion fluids of the in vitro digestion model (per 1000 mL) (Versantvoort et al. 2005; Walczak et al. 2012).

Saliva (pH 6.8 ± 0.1)	Gastric fluid (pH 1.3 ± 0.1)	Duodenal fluid (pH 8.1 ± 0.1)	Bile fluid (pH 8.2 ± 0.1)
896 mg KCl 200 mg KSCN 1021 mg NaH2PO4·H2O 570 mg Na2SO4 298 mg NaCl 1694 mg NaHCO3 200 mg urea 290 mg amylase 15 mg uric acid 25 mg mucin Nano-pure water	2752 mg NaCl 306 mg NaH2PO4·H2O 824 mg KCl 302 mg CaCl2 306 mg NH4Cl 6.5 mL 37% HCl 650 mg glucose 20 mg glucuronic acid 85 mg urea 330 mg glucosamine-hydrochloride 1 g BSA 2.5 g pepsin 3 g mucin Nano-pure water	7012 mg NaCl 3388 mg NaHCO3 80 mg KH2PO4 564 mg KCl 50 mg MgCl2·6H2O 180 mL HCl (37%) 100 mg urea 151 mg CaCl2 1 g BSA 9 g pancreatin 1.5 g lipase Nano-pure water	5259 mg NaCl 5785 mg NaHCO3 376 mg KCl 150 mL HCl (37%) 250 mg urea 167.5 mg CaCl2 1.8 g BSA 30 g bile Nano-pure water Sodium carbonate solution 84.7 g NaHCO3 Nano-pure water

Table 2. Physicochemical characteristics of AgNPs.

Medium	Incubation time (h)	Hydrodynamic diameter (nm) ± SD		ζ-potential (mV) ± SD
		(LA) AgNPs	(Cit) AgNPs	(LA) AgNPs	(Cit) AgNPs
Nano-pure water	0	56 ± 8	51 ± 1	−56 ± 10	−37 ± 7
	24	49 ± 2	48 ± 4	−36 ± 10	−29 ± 12

Figure 1. Silver content in pristine and in vitro digested (IVD) AgNPs suspensions and AgNO₃ solution. (a) Silver content measured as total Ag using ICP-MS. (b) Silver content measured as AgNPs using spICP-MS. Concentrations are given as the mean ± SD (n = 3). *Significant difference between the pristine and IVD suspension of the same AgNPs (p < 0.05).

Figure 2. Cell viability of a Caco-2/HT29-MTX co-culture after 24 h exposure to a concentration range of pristine: (a) (LA) and (Cit) AgNPs and (b) AgNO₃, and IVD; (c) (LA) and (Cit) AgNPs and (d) AgNO₃ using the WST-1 viability assay. Viability is given as a percentage of the control (mean ± SD; n = 3). ^×Significant difference between AgNPs at the same concentration (p < 0.05). ^*,#Significant difference between any concentration and the control within the same treatment (p < 0.05).

Figure 3. Silver uptake/adhesion of (LA) and (Cit) AgNPs and AgNO₃ in differentiated Caco-2/HT29-MTX cells after 24 h exposure, measured and expressed as total Ag after (a) exposure to pristine AgNPs and AgNO₃; (c) exposure to in vitro digested AgNPs and AgNO₃. Also expressed as AgNPs after (b) exposure to pristine AgNPs and AgNO₃; (d) exposure to in vitro digested AgNPs and AgNO₃. Concentrations are given as the mean ± SD (n = 3). Values with different letters are significantly different at the same concentration (p < 0.05).

Figure 4. Uptake/adhesion of AgNPs after 24 h exposure. Confocal microscopy images of 21-day-old Caco-2/HT29-MTX cells. (a) without exposure to AgNPs (negative control), after exposure to 2500 μg/L, (b) pristine (LA) AgNPs, (c) pristine (Cit) AgNPs, (d) IVD (LA) AgNPs, and (E) IVD (Cit) AgNPs. Nuclei were stained in red (RedDot-2), actin was stained in green (Alexa-488 Phalloidin) and AgNPs are shown in white (back scatter).

Figure 5. Number-weighted size distributions generated by spICP-MS measurements in exposure suspensions. (a) 500 µg/L pristine (LA) AgNPs, (b) 500 µg/L pristine (Cit) AgNPs, (c) 250 µg/L pristine AgNO₃, (d) (LA) 500 µg/L IVD AgNPs, (e) 500 µg/L IVD (Cit) AgNPs, and (f) 250 µg/L IVD AgNO₃.

Figure 6. Number-weighted size distributions generated by spICP-MS measurement of Caco-2/HT29-MTX cellular silver content after 24 h exposure to (a) 500 µg/L pristine (LA) AgNPs, (b) 500 µg/L pristine 50 nm (Cit) AgNPs, (c) 250 µg/L pristine AgNO₃, (d) 500 µg/L IVD (LA) AgNPs, (e) 500 µg/L IVD (Cit) AgNPs, and (f) 250 µg/L IVD AgNO₃.

Versantvoort, C. H. M., A. G. Oomen, E. Van de Kamp, C. J. M. Rompelberg, and A. J. A. M. Sips. 2005. “Applicability of an In Vitro Digestion Model in Assessing the Bioaccessibility of Mycotoxins from Food.” Food and Chemical Toxicology 43(1): 31–40. doi:10.1016/j.fct.2004.08.007.

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Walczak, A. P., R. Fokkink, R. Peters, P. Tromp, Z. E. Herrera Rivera, I. M.C.M. Rietjens, P. J.M. Hendriksen, and H. Bouwmeester. 2012. “Behaviour of Silver Nanoparticles and Silver Ions in an In Vitro Human Gastrointestinal Digestion Model.” Nanotoxicology 7(7): 1198–1210. doi:10.3109/17435390.2012.726382.

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