Acknowledgment
Cornelia Voigtlọnder gratefully acknowledges funding of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) by the German Research Foundation (DFG) in the framework of the German excellence initiative. This work was supported by the project prepara- tory fund of the University of Erlangen-Nürnberg.
References
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Chapter 10
Screening Applications to Test Cellular Fitness in Transwell® Models After Nanoparticle Treatment
Bastian Christ*, Christina Fey*, Alevtina Cubukova, Heike Walles, Sofia Dembski, and Marco Metzger
Abstract
Nanoparticles (NPs) in biotechnology hold great promise for revolutionizing medical treatments and therapies. In order to bring NPs into clinical application there is a number of preclinical in vitro and in vivo tests, which have to be applied before. The initial in vitro evaluation includes a detailed physicochemical characterization as well as biocompatibility tests, among others. For determination of biocompatibility at the cellular level, the correct choice of the in vitro assay as well as NP pretreatment is absolutely essential.
There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advan- tages and disadvantages. Regardless of the assay method chosen, the major factors critical for reproduc- ibility and success include: (1) choosing the right assay after comparing optical NP properties with the read-out method of the assay, (2) verifying colloidal stability of NPs in cell culture media, (3) preparing a sterile and stable NP dispersion in cell culture media used in the assay, (4) using a tightly controlled and consistent cell model allowing appropriate characterization of NPs. This chapter will briefly summarize these different critical points, which can occur during biocompatibility screening applications of NPs.
Key words Nanoparticles, Cytotoxicity assay, Impedance spectroscopy, Colloidal dispersion of nanoparticles
1 Introduction
Well-tailored multifunctional nanoparticles (NPs) play a major role in the development of future-oriented advanced functional materi- als for life science applications. The surface engineering and appli- cation of NPs in biotechnology holds great promise for revolutionizing medical treatments and therapies in areas such as imaging, faster diagnostics, drug delivery, and tissue regeneration.
Because of the NP size, which is similar to subcellular structures such as proteins or DNA, they can interact with each other and
*Bastian Christ and Christina Fey contributed equally to this work.
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initiate various processes at this level. Despite the already existing diversity of R&D work on the NP field, there is still reinforced need to study NP clinical relevant properties and to bring them into the application. One of the biggest challenges here is the cor- relation of physicochemical NP properties such as size, morphol- ogy, and surface quality and their in vivo interactions [1].
More and more new smart NP systems are synthesized. There are numerous methods to investigate physicochemical properties of novel NP. The NP composition (qualitative and quantitative) can be determined using wet chemical analysis, mass spectrometry with inductively coupled plasma (ICP), and X-ray fluorescence (XRF). The dynamic light scattering (DLS) and the Fraunhofer diffraction studies allow the determination of particle size and polydispersity. More information on the particle size and mor- phology can be obtained by the transmission (TEM) and scanning electron microscopy (SEM). Another method, applied in the characterization of NPs, is X-ray diffraction (XRD). Thus, crystal structure and size can be determined. Usually, there are different spectroscopic methods such as nuclear magnetic resonance (NMR), infrared (IR) and Raman spectroscopy, as well as deter- mination of the surface charge (Zeta-potential) available for sur- face characterization. The biggest challenge is the quantitative determination of the surface features. NP surface can be charac- terized by thermogravimetry (TGA), elemental analysis (CHNS), X-ray photoelectron spectroscopy (XPS), and potentiometric and photometric techniques. These methods, established in the sur- face analysis as standard, should be adapted and calibrated for the NP characterization.
The methods of NP biocompatibility estimating are also imperfect. For a reliable in vitro determination of pathophysiologi- cal effects at the cellular level, the correct choice of the method of examination as well as NP pretreatment is absolutely essential. The method must be selected so that there are no artifacts, otherwise false-positive or -negative results will be achieved. Different tests can be performed to evaluate the cytotoxicity of NP (cell growth and cell survival): the NADH/NADPH-dependent MTT test [3-(4.5-dimethyl-thiazol-2-yl)-2.5-diphenyl-tetrazolium bromide], WST-1 test [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2.4- disul- phophenyl)2H-tetrazolium], and eluation test according to DIN EN ISO 10993-5 or luminescent ATP-dependent tests such as CellTiter-Glo® from Promega. Before examination, it is important to choose the appropriate test depending on the material system and further NP application. For example, it is crucial to note that the read-out of some methods is based on luminescence (e.g., CellTiter-Glo®). In the case of fluorescence dye-doped NP systems, the influence of NP absorption and emission properties on the detection and following results must be excluded. A quite promis- ing and complementary nondestructive possibility to determine Bastian Christ et al.
cell fitness after NP application is the impedance spectroscopy or transepithelial electrical resistance (TEER) measurement. In con- trast to standard testing procedures, these methods allow compari- son of individual tissue models before and after a treatment [2].
Interestingly, also mild effects, e.g., by repeated washing steps with PBS already led to reduced electrical resistance in skin models, which later recovered indicating the high sensitivity of this tech- nique. Furthermore, MTT-based assays, for instance, assess only the viable cells, thus nonviable cell layers such as present in epider- mal models are not considered, however these cells pose the major part of the skin barrier. In conclusion, impedance spectroscopy could be useful to identify sub-irritative (e.g., stinging, burning, or itching sensation) or mild chemical effects on barrier integrity, for instance.
With respect to NP applications, the next important aspect is the NP stability under examination conditions. Without appropri- ate surface modification, NPs tend to agglomerate and sediment in physiological solutions due to their pH and salt content [3–5].
This needs to be addressed in order to ensure a consistent assess- ment of the therapeutic and diagnostic potential of NPs in cell culture experiments or animal testing: studies concerning particle concentrations are only reliable when carried out with stable dis- persions that guarantee an accurate dosing. The effect of different sized particles on cells can only be studied if the particles do not form large agglomerates [3, 6, 7]. The agglomeration leads to an unwanted reduction of surface area and therefore falsifies results [3, 8]. Consequently, choosing the right stabilizing agent is of sig- nificant importance. However, the efficiency of a stabilizer depends not only on the NP composition but also on its surface properties and the chosen cell culture medium [5]. Therefore, since there is no general solution, the best surfactant has to be identified in each individual particle case and medium [9].
2 Materials
1. 70% Ethanol solution.