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. 2011 Feb 9;8(1):8.
doi: 10.1186/1743-8977-8-8.

Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects

Gertie J Oostingh  1 , Eudald Casals, Paola Italiani, Renato Colognato, René Stritzinger, Jessica Ponti, Tobias Pfaller, Yvonne Kohl, Daniëlla Ooms, Flavia Favilli, Hilde Leppens, Davide Lucchesi, François Rossi, Inge Nelissen, Hagen Thielecke, Victor F Puntes, Albert Duschl, Diana Boraschi
Affiliations

Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects

Gertie J Oostingh et al. Part Fibre Toxicol. .

Abstract

Background: With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated.

Results: In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin.

Conclusion: The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

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Figures

Figure 1
Figure 1
Absorption spectra of NPs. Upper panel: UV-visible spectra of Ag NPs (30 nm; a, red colour) and Au NPs (10 nm; b, green colour) exposed to cell culture medium (DMEM + 10% FBS) at 37°C. Fainter lines: NPs as synthesized. Darker lines: after 48 h in culture medium at 37°C. Red shift of the absorbance peak is an indication of protein corona formation. Lower panel: absorption spectra of the NPs used in this work. NP concentration is 1 × 1012 NPs/ml for Au, Ag and AgO and 1 × 1014 NPs/ml for CeO2, CoO and Fe3O4. At these concentrations, all NPs absorb in the visible range, where most of the biological tests have their readouts. In this figure: 340 nm is the absorption wavelength of NADH, measured in the LDH assay; 405 nm is the absorption wavelength of the chromophore p-nitroaniline (pNA) measured in the LAL assay for endotoxin determination; 440 nm, 490 nm and 540 nm are the readout for WST-1, MTS and MTT respectively.
Figure 2
Figure 2
TEM images and size distribution analysis of NPs exposed to different aqueous media. Upper panels: 13 nm Au NPs: A, as synthesized; B, after 48 h incubation at 37°C with DMEM + 10% FBS. Lower panels: 7 nm CoO NPs: C, as synthesized in dichlorobenzene; D, after 24 h at room temperature after phase transfer to water. Scale bars are 100 nm.
Figure 3
Figure 3
Suitability of different cell viability assays for nanotoxicity testing. Cytotoxicity of different NPs (suspended from dry powders) on THP-1 cells was tested after 48 h incubation using the Cytotox-96 assay from Promega (left panel) and the Toxilight assay from Lonza (center panel). In addition, the CellTiter-Blue assay from Promega was used to determine the cell viability (right panel). Data were normalised to the PBS control (9.1% v/v PBS) and mean values ± SD are shown. The dotted lines represent the values in control cells with PBS (taken as 0% cytotoxicity in left and centre panels, and as 100% viability in the right panel). The shaded areas represent the SD above and below the control values. * p < 0.05 vs. untreated control.
Figure 4
Figure 4
Solvent immunotoxicity. Four different particle solvents were tested for their immunomodulation on IL-6 promoter transfected A549 cells, either unstimulated or stimulated with TNF-α (20 ng/ml), after 48 h. For IL-6 promoter induction, the luminescence value of untreated cells was 220 ± 23 RLU (relative light units), while TNF-α-stimulated cells had a value of 1,485 ± 211 RLU. Data were normalised to allow a direct comparison between stimulated and unstimulated cells and to enable combining data from multiple experiments. * p < 0.05 vs. untreated control.
Figure 5
Figure 5
Production of inflammation-related soluble factors by CaCo-2 cells. Cells were chronically exposed for 15 days to culture medium alone or containing IL-1β (10 ng/ml, positive control), Au NPs 4 nm (5.2 μg/ml), CoO NPs (3.5 μg/ml), CeO2 NPs (1.3 μg/ml) (all corresponding to 9.1% v/v), or their solvents. Factors were detected in the cell supernatants by Proteome Profiler Antibody Array and evaluated as arbitrary units by densitometric analysis. Results for IL-1β, sICAM-1, CXCL1 and CXCL4 are reported in the figure, and presented as mean values ± SD from 2-4 replicate determinations.
Figure 6
Figure 6
Measuring endotoxin contamination in NP preparations. As preliminary step, evaluation of the interference of Au NPs on the pNA readings at 405 nm was performed. Different concentrations of Au NPs (4, 13, and 20 nm diameter) could significantly increase the readout at OD405 of 125 μM pNA (round symbols) (p < 0.05 for all concentrations of NPs 4 nm, for the three highest of NPs 13 nm, and for the highest of NPs 20 nm) (upper left panel). The selected pNA concentration corresponds to that developed by 0.6 EU of endotoxin in the Endpoint Chromogenic LAL assay (lower left panel). The increase caused by NPs could therefore be misinterpreted as a significant increase in the presence of endotoxin. For this reason, endotoxin evaluation was then performed only on NP dilutions that did not cause significant interference with the pNA readings (typically, ≤ 1 μg/ml for Au NPs 4 nm, ≤ 4 μg/ml for Au NPs 13 nm, and ≤ 12.5 μg/ml for Au NPs 20 nm). Five separate batches of Au NPs 4 nm and their solvents were tested. For dry NPs, the solvent was endotoxin-free PBS. Batch-to-batch variability in the endotoxin contamination was evident (upper right panel). The importance of avoiding such contamination is shown by the powerful effect of minute amounts of endotoxin in activating IL-1β gene expression in human monocytes (lower right) (p < 0.05 for all endotoxin concentrations vs. control; square symbols).
Figure 7
Figure 7
NP-induced IL-8 promoter activation in A549 cells. Four different NP powders were used to test their immunomodulatory effect on A549 cells, measured by the induction of the IL-8 promoter in transfected cells. Unstimulated cells (diamond shaped symbols) or 20 ng/ml TNF-α stimulated cells (square symbols) were exposed to increasing concentrations of NPs for 48 h. Mean values ± SD (n = 3) are shown. The dotted lines represent the values in control cells treated with PBS (100%). The shaded areas represent the SD above and below the control values. Untreated cells gave a luminescence value of 2,799 ± 367 RLU, while TNF-α-stimulated cells had a value of 100,366 ± 3,697 RLU. Data were normalised to the PBS control (9.1% v/v PBS; taken as 100% value) to allow a direct comparison between stimulated and unstimulated cells and to enable combining data from multiple experiments. * p < 0.05 vs. control
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