Multiplexing techniques for measurement of the immunomodulatory effects of particulate materials: Precautions when testing micro- and nano-particles
Graphical abstract
An illustration of some of the complexities with the accuracy of multiplex assay measurement of biomarker expression in response to in vitro or in vivo exposure to micro- or nano-particles.
Introduction
Nanoparticles have widespread pharmaceutical and clinical applications, where safety and interactions with biomolecules are of critical significance [1], [2], [3]. The type and composition of nanomaterials play a pivotal role in modulating the immune system, which may compromise therapeutic outcomes and can be life-threatening, in particular, when the immunomodulation is unintentional [4], [5]. Advances in multiplexing techniques and polychromatic flow cytometry have enabled the profiling of several hundreds of biomarkers in a few microliter sample volumes, with several existing limitations [6]. Multiplexing assays for evaluating immunotoxicity of these biomaterials are usually performed using the same approach utilized for small molecules. However, there are key differences in the physicochemical and biological characteristics between small molecules and large/assembled structures, which should not be overlooked [7], [8]. In particular, the ability of assembled structures and particles to adsorb biomarkers on their surfaces and/or sequester them into their internal domains, in contrast to small molecules, is one of the main obstacles that may influence the accuracy of the multiplexing strategies for quantitative assessment of the expression of biomarkers after treatment with particulate materials. We have recently reported on the necessity for reassessment of immunotoxicity of nanomaterials, and some important precautions to account for the differences in behavior between assembled/macromolecular structures and small molecules [7]. The experimental details and precautions described herein can allow for precise evaluation of biomarker expression in response to particulate materials, pharmaceuticals and medical devices, and should be taken into careful consideration in pre-clinical and clinical studies, and other processes that involve the use of multiplexing techniques.
Section snippets
Multiplexing technique for evaluation of particulate materials
There are several strategies that can be utilized for evaluation of biomarkers in response to treatment with various biological agents. For instance, aggregation-induced emission-active functional materials have been extensively used for fluorescence sensing and biological imaging, and thus could be utilized as fluorescence signals for biomarkers detection [9], [10], [11]. However, a focus is being given here on the use of multiplexing technique as an efficient and commonly exploited strategy
Pre-assessment step: optional
Biomarkers may be adsorbed on the surfaces and/or absorbed within internal regions of micro- or nano-particles [4], [12], [13], [14], [15]. This interference reduces the available concentrations of the biomarkers, thereby, leading to potential artifacts and data misinterpretation (Fig. 6). The extent of interference depends on the nature of biomarker and the physicochemical characteristics of the tested materials. Recently, we have reported on the necessity for reassessment of nanomaterials
Data mining and analysis
Data output are usually presented as median fluorescence intensity (MFI) and concentration (pg/mL). The outliers should be removed prior to data analysis. This can be done manually or automatically depending on the software. Negative values might be due to a lower amount (or absence) of analyte or due to an effect of the sample matrix. Remember that the blank values are usually subtracted automatically from the resultant values of the all other assay wells.
Competing interests statements
The authors declare no competing financial interests.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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