Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects
Particle and Fibre Toxicology
Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects
Gertie J Oostingh 0
Eudald Casals
Paola Italiani
Renato Colognato
Ren Stritzinger 0
Jessica Ponti
Tobias Pfaller 0
Yvonne Kohl
Danilla Ooms
Flavia Favilli
Hilde Leppens
Davide Lucchesi
Franois Rossi
Inge Nelissen
Hagen Thielecke
Victor F Puntes
Albert Duschl 0
Diana Boraschi
0 Department of Molecular Biology, University of Salzburg , 5020 Salzburg , Austria
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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Background
The potential benefits and the risks associated with the
application of nanomaterials have been widely debated
in recent years. The need to correctly assess
nanoparticle (NP) risks in order to protect workers, consumers
and the environment is well accepted in the scientific
and regulatory community [1,2]. Both the human
population and the environment may be exposed to
nanomaterials during all stages of the NP life cycle: raw material
production, transport and storage, industrial use,
consumer use, and waste disposal. The consumer use
can vary from products like coated textiles or paints,
where the presence of nano-products is not clearly
stated, to sunscreens, where the NP content is explicitly
labelled. In addition, medical use of NPs for diagnostic
purposes or as drug delivery backbone represents
intentional exposure to significant NP doses. Currently, a
variety of methodologies are being discussed and
evaluated to perform a complete risk assessment of
nanomaterials. There are a number of European legislations that
have the objective of implementing laws regarding use
of and exposure to nanomaterials [3,4] including the
REACH programme [5]. However, a lack of information
on exposure levels, in vitro and in vivo NP effects and
the life cycle of these entities make implementation of
standards extremely difficult.
Even though a wealth of publications addresses the
delicate issue of toxicity of engineered NPs [1,6,7], the
exact events that occur in the interaction between NPs
and the immune system are still largely unknown, even
though nanoparticle-induced alterations of the immune
system can have important effects on human health [8].
Despite a worldwide effort, results are overall
contradictory, in particular when (immuno-) toxicity of NPs
in vitro or in vivo is concerned, and no clear-cut
information can be provided to the policy-makers, the
producers and workers, and the public at large. Results
obtained in different laboratories can often not be
compared because of a lack of disclosure of experimental
details as well as a lack of standardisation of methods
and reagents.
An important aspect is that nanoparticle
characterisation should also be performed at the point of use, since
ageing, storage conditions and contamination can
modify their properties in important ways. Alterations in
particle characteristics can also occur when
nanomaterials get in contact with the human body or with
biological entities in the environment. Biological molecules can
modify the nanomaterials and cause dissolution,
aggregation or, at the very least, coating. The result can be
anything from free ions or chemicals released from
nanomaterials to micrometer-sized aggregates. Coating,
for example by polysaccharides or proteins, may render
the materials less harmful but can also change their
properties in unexpected ways [9,10]. Furthermore,
association with biological molecules such as endotoxins,
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