Data supporting the investigation of interaction of biologically relevant proteins with ZnO nanomaterials: A confounding factor for in vitro toxicity endpoints

Test materials, like manufactured nanomaterials (MN), may interact with serum proteins, interleukins (IL) and lactate dehydrogenase (LDH) and cause measurement artefacts as a result of e.g., physical adsorption and electrostatic forces, and/or interaction with dissolved species or conditional chemical changes during testing. In this article, data are given on the zeta-potentials of two manufactured ZnO nanomaterials (NM-110 and NM-111) dispersed in 0.05% w/v Bovine Serum Albumin (BSA) water batch dispersions and in Ham's F12 nutrient mixture added Fetal Bovine Serum (FBS), penicillin, and streptomycin and particle free mediums (cHam's F12). Data on the Zeta-potential and the iso-electrical point of lactate hydrogenase in pure Ham's F12 nutrient mixture is also provided. The percentage of added IL-6, IL-8 and LDH remaining after 24-h incubation in cHam's F12 are given as function of MN concentrations. Finally data from thermodynamic chemical reaction modeling of changes in pH and Zn-speciation during dissolution of ZnO or dissolved ZnCl2 additions to Ham's F12 using Geochemist Workbench® are given. For further information, data interpretation and discussion please refer to the research article “Interaction of biologically relevant proteins with ZnO nanomaterials: a confounding factor for in vitro toxicity endpoints” (E. Da Silva et al. 2019).


a b s t r a c t
Test materials, like manufactured nanomaterials (MN), may interact with serum proteins, interleukins (IL) and lactate dehydrogenase (LDH) and cause measurement artefacts as a result of e.g., physical adsorption and electrostatic forces, and/or interaction with dissolved species or conditional chemical changes during testing. In this article, data are given on the zeta-potentials of two manufactured ZnO nanomaterials (NM-110 and NM-111) dispersed in 0.05% w/v Bovine Serum Albumin (BSA) water batch dispersions and in Ham's F12 nutrient mixture added Fetal Bovine Serum (FBS), penicillin, and streptomycin and particle free mediums (cHam's F12). Data on the Zeta-potential and the iso-electrical point of lactate hydrogenase in pure Ham's F12 nutrient mixture is also provided. The percentage of added IL-6, IL-8 and LDH remaining after 24-h incubation in cHam's F12 are given as function of MN concentrations. Finally data from thermodynamic chemical reaction modeling of changes in pH and Zn-speciation during dissolution of ZnO or dissolved ZnCl 2 additions to Ham's F12 using Geochemist Workbench ® are given. For further information, data interpretation and discussion please refer to the DOI   .

Value of the data
The surface charge of MN and the adsorption of serum proteins from the dispersion medium on to ZnO MN are portrayed. This is important because these parameters can affect MN fate and interaction with specific protein assay 1À6 It is important to account for interaction between the MN and measured biomolecules, e.g. interleukins, LDH, lipids, and reactive oxygen species, which could lead to artefacts in the identification and quantification of biological response The value of thermodynamic chemical reaction modeling is demonstrated and show that an increase in dissolved Zn from ZnO MN in cHAM's F12 can lead to an increase in pH, reducing the negative surface charge towards neutral Parameters that may be determinants for LDH interaction (Zn-ion concentration due to dissolution of ZnO MN in the test medium, changes in pH, and changes in protein zeta potentials) can be shown to vary during incubation 320 and 640 mg ZnO/mL and the 0.05% w/v BSA water batch dispersions with 2560 mg ZnO/mL. The zeta potentials of the particle free mediums are shown for reference. The pH values in the dispersions were close to neutral (NM-110: pH ¼ 7.0 ± 0.3; NM-111: pH ¼ 6.8 ± 0.3) during measurements. Table 2 lists the percentage of the initially added levels of IL-6 (500 pg/mL), IL-8 (4000 pg/mL) and LDH (100 ng/mL) after incubation with NM-110 and NM-111 at eight different doses (0e640 mg/mL) in cHam's F12: The LDH data (OD LDH ) are given as the optical density for LDH expressed as a percentage of optical density of the medium reference OD ref without MN. IL-6 and IL-8 concentrations are given as the percent of the initially dosed concentrations. Fig. 1 shows the zeta-potential data as function of pH and the iso-electrical point of LDH at pH ¼ 4.67 (vertical line, triplicates) in pure Ham's F12 nutrient mixture. A concentration of 10000 ng LDH/mL was used to allow good zeta-potential measurements. 1 M HCl and 1 M NaOH were used for pH-titrations. Fig. 2 shows the pH-variation as function of the amount of ZnO ( Fig. 2A and B) and ZnCl 2 ( Fig. 2C and D) reacted and as function of Zn-concentrations in Ham's F12 nutrient mixture calculated using the React program in the Geochemist Workbench ® [3]. The pH may increase to approximately 9.5 during dissolution of only 10 mg ZnO/mL ( Fig. 2A and B). Opposite, a slight reduction in pH from pH 7.4 to pH 6.7 occurs with increased amounts of dissolved ZnCl 2 ( Fig. 2C and D). Fig. 3 shows the data from calculations of the Zn speciation in Ham's F12 nutrient mixture during dissolution of ZnO and ZnCl 2 . More than 99.3% (by weight) of the dissolved Zn will occur as Zn 2þ during dissolution of ZnO (Fig. 3A). Slightly less (>95.8% by weight) will be Zn 2þ during dissolution of ZnCl 2 (Fig. 3B).  sd ¼ standard deviation, obtained from three independent experiments with two replicates each. a Denotes a statistically significant difference with the baseline (ANOVA, p-value <0.001).   Batch dispersions of the test materials were produced by dispersing 2.56 mg/mL in sterile filtered 0.05% (v/v) BSA water (Nanopure Diamond UV) by probe-sonication as described in the NANO-GENOTOX batch dispersion protocol [5]. Sonication was performed using a Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) operated for 16 min at 400 W and 10% amplitude using a 13 mm disruptor horn. For further details please see Jensen et al. [5].
The complete test medium (cHam's F12) consisted of Ham's F12 nutrient mixture (ThermoFisher Scientific, Hvidovre, Denmark) with 1% v/v penicillin/streptomycin (10000 U/mL and 10 mg/mL respectively; In Vitro A/S, Fredensborg, Denmark) and 10% v/v of inactivated Fetal Bovine Serum (USA grade; In Vitro A/S, Fredensborg, Denmark). For interaction studies cHam's F12 was added 500 pg/mL IL-6 standard (National Institute for Biological Standards and Controls, Hertforshire, UK), 4000 pg IL-8 standard (National Institute for Biological Standards and Controls, Hertforshire, UK; final concentration), and 100 ng/mL rabbit muscle LDH (Sigma-Aldrich, Brøndby, Denmark IL-6, IL-8 and LDH levels in cHam's F12 with and without test materials were determined after 24 h incubation in darkness at 37 C and a 5% CO 2 atmosphere with a 95% relative humidity in a cell incubator (CelCulture ® CO 2 Incubator, ESCO Medical, Egaa, Denmark) maintaining a temperature of 37 C and a 5% CO 2 atmosphere with a 95% relative humidity in the dark without shaking. Suspensions were collected, centrifuged and prepared for spectrophotometric analysis of interleukine (BD Pharmingen kit, Cat. No. 555220 and 555244 respectively; BD Biosciences, Lyngby, Denmark) and LDH (Roche Diagnostics GmBH, Roche Applied Science, Mannheim, Germany) using an ELISA reader as described in Da Silva et al. [1].
Thermodynamic chemical reaction modeling of dissolved Zn concentration associated effect on pH and Zn-ion speciation after addition of ZnO and ZnCl 2 was completed using the Geochemist Workbench ® v. 11.0 [3]. Please see Da Silva et al. [1] for further details on medium composition and conditions defined for generating the data.
Statistical analysis, tables and graphs were made using R (version 3.4.3) and Gt-plot in Geochemist Workbench ® v. 11.0. Statistical significance was set at p-value < 0.05. Each treatment was repeated in duplicate and each experiment replicated three times.