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

Oostingh GJ, Casals E, Italiani P, Colognato R, Stritzinger R, Ponti J, Pfaller T, Kohl Y, Ooms D, Favilli F, Leppens H, Lucchesi D, Rossi F, Nelissen I, Thielecke H, Puntes VF, Duschl A, Boraschi D - Part Fibre Toxicol (2011)

Bottom Line: To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria. geja.oostingh@sbg.ac.at

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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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.
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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.

Mentions: NPs, as colloidal particles, are systems obtained from a chemical equilibrium, and exist in a meta-stable phase. Their final fate is the disintegration or agglomeration towards more stable phases [37]. Thus, even when the PC prevents the NPs from aggregation, the particles can still corrode. We have observed that all the inorganic NP preparations that were used in this work showed a release of cations with time (data not shown). Similar data were published for quantum dots (CdSe) [38] and carbon nanotubes [39], which corrode and release toxic Cd ions and less toxic carbon derivatives, respectively. Another consequence that should not be neglected, given the importance of size in the nano-bio interaction, is that corroded particles are much smaller in size compared to the original material. Striking evidence of this phenomenon is provided by the TEM images of Co NPs, showing that the morphology of the particles is severely affected 24 h after their dispersion in water (Figure 2). Spherical NPs with a homogeneous crystal contrast are transformed into shapeless NPs with a broader size distribution and polycrystalline nature. Together with the morphological transformation, an increase of Co ions in solution has been observed by inductively-coupled plasma mass spectrometry (up to 10% of the total NP mass; data not shown). This process is concomitant with the oxidation of Co towards CoO, a phenomenon that has been observed for all NPs. From the TEM images, a dissolution of 13% of the mass can be estimated for Co NPs incubated for 24 h at room temperature after phase transfer from just synthesized in dichlorobenzene to water, and of 11% of the mass for Au NPs after 48 h in cell culture medium (DMEM + 10% FBS) at 37°C.


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

Oostingh GJ, Casals E, Italiani P, Colognato R, Stritzinger R, Ponti J, Pfaller T, Kohl Y, Ooms D, Favilli F, Leppens H, Lucchesi D, Rossi F, Nelissen I, Thielecke H, Puntes VF, Duschl A, Boraschi D - Part Fibre Toxicol (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045340&req=5

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.
Mentions: NPs, as colloidal particles, are systems obtained from a chemical equilibrium, and exist in a meta-stable phase. Their final fate is the disintegration or agglomeration towards more stable phases [37]. Thus, even when the PC prevents the NPs from aggregation, the particles can still corrode. We have observed that all the inorganic NP preparations that were used in this work showed a release of cations with time (data not shown). Similar data were published for quantum dots (CdSe) [38] and carbon nanotubes [39], which corrode and release toxic Cd ions and less toxic carbon derivatives, respectively. Another consequence that should not be neglected, given the importance of size in the nano-bio interaction, is that corroded particles are much smaller in size compared to the original material. Striking evidence of this phenomenon is provided by the TEM images of Co NPs, showing that the morphology of the particles is severely affected 24 h after their dispersion in water (Figure 2). Spherical NPs with a homogeneous crystal contrast are transformed into shapeless NPs with a broader size distribution and polycrystalline nature. Together with the morphological transformation, an increase of Co ions in solution has been observed by inductively-coupled plasma mass spectrometry (up to 10% of the total NP mass; data not shown). This process is concomitant with the oxidation of Co towards CoO, a phenomenon that has been observed for all NPs. From the TEM images, a dissolution of 13% of the mass can be estimated for Co NPs incubated for 24 h at room temperature after phase transfer from just synthesized in dichlorobenzene to water, and of 11% of the mass for Au NPs after 48 h in cell culture medium (DMEM + 10% FBS) at 37°C.

Bottom Line: To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria. geja.oostingh@sbg.ac.at

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.

Show MeSH
Related in: MedlinePlus