Drug Delivery to the Lungs, Volume 33, 2022 – Altin Kocinaj et al

In-Vitro modelling of the lungs response to nanoparticulates

Altin Kocinaj1, Laura Urbano1, Darragh Murnane1

1
University of Hertfordshire, College Lane, Campus, Hatfield AL10 9AB

Summary

Nanoparticles are abundant in everyday life, with air pollution and consumer products leading to a
growing number of concerns for potential health effects of inhaled exposure. There is no standalone
alternative for animal toxicity studies, thus we sought to build a predictive cell culture model using high
content imaging/analysis (HCA) and traditional viability assays. Human lung epithelial cells (NCI-H441)
were seeded for in a 96-well plate and exposed to a panel of nanoparticles for 24 hours, for high
content imaging. For the HCA, plated cells were given fluorescent labels which allowed both
morphological assessment (such as cell area and diameter, membrane permeability and mitochondrial
activity) and cell segmentation for the consequent analysis. Image acquisition was performed using an
automated fluorescence imager (EVOS M7000) and was segmented and analysed using the
accompanying Celleste program. Morphological differences can be seen and analysed using HCA,
with the possible identification of subgroups of cells. There are differences in such parameters as cell
area with ~100% of cells exposed to 33ug/ml of copper oxide being below the median cell area of the
control or lowest concentration used. Also, a flattening and spread of the 33ug/ml distribution
indicating a larger population of cells compared to control with greater membrane permeability. These
preliminary results indicate the possible use of HCA to define morphometric cell-by-cell changes and
population subgroups that are not able to be elucidated by a single value from standard assays (such
as Prestoblue – cell viability).

Key message

The use of inexpensive cell culture techniques coupled with the use of HCI/A (high content
imaging/analysis) can provide the means of identifying morphometric changes within the cell which
would otherwise be ignored. When combined with biochemical assays and expression studies enables
a potentially more complete toxicological picture.

Introduction

Nanoparticles are particles in the range of 1-100nm having at least one dimension less than 100 nm
and comprise a class of materials that exhibit unique physical, chemical, and biological properties,
differing distinctly from their subsequent small molecules and bulk materials(1). Human exposure is
inevitable with the abundance of everyday consumer products and the environmental release of
nanoparticles. What is evident, is the poor understanding and investigation of the health risks
nanoparticles may pose both in acute and chronic exposure (1).

Particulate matter describes the sum of the particles both solid and liquid which are suspended in air
and can penetrate into the respiratory tract. Nanoparticles have also revolutionised the state of
biomedicine due to their unique properties (such as systemic stability, level of solubility, and target site
specificity) in the field of drug delivery. As exceptional as the benefits are, these particles have the
potential to be damaging through our limited knowledge of toxicity, and non-specific protein
interactions thus highlighting the importance of nanotoxicology studies(2).

For toxicological studies, animal models remain the gold standard, and there are no standalone
alternatives in representing human lung toxicity interactions. They have provided invaluable data
regarding compound toxicology. However, historically the translation into human subjects from animal
models is particularly unpredictable. Thus, it has been brought into question over recent years due to
ethical concerns and more importantly its clinical application and validity(3). This has brought about
studies of the predictability of animal studies for replicating within human clinical trials which based on
this review showed only 37% were replicated in humans and 20% were contradicted when tested in
humans(4).

Within toxicology experimentation of inhalable substances cell culture has been used to determine the
toxicity of the compounds of interest, however much like the animal models they prove to be a poor
Drug Delivery to the Lungs, Volume 33, 2022 – In-Vitro modelling of the lungs response to
nanoparticulates
representation of the human lung and its interaction with the compounds(5). This is due to such
limitations as single cell type structure not allowing representative cell function given by differentiated
cell phenotype cross-talking. Additionally, submerged conditions of 2D cultures do not represent the
lungs and can cause agglomeration of the particles when testing thus not allowing for the testing of the
correct size (possibly giving a different toxic reaction)(5). However, they do provide many benefits
such as being inexpensive and time efficient in comparison to animal models(6). There is still a gap
between cell culture models and animal models in terms of toxicity representation, however they may
provide a more efficient and inexpensive form of toxicological analysis of particle of interest both for air
pollution concerns or pharmaceutical development.

Bridging the gap between animal models, with cell culture and ultimately human trials this study aims
to develop a standalone cell culture model for this purpose. Encompassing the use of 2D cell culture,
high content imaging and analysis to give us a more detailed account and understanding of the
morphological changes of individual cells exposed to known toxic nanoparticles.

Experimental methods

Cell culture

Human lung epithelial cell line (NCI-H441) was cultured in RPMI 1640 medium with 10% v/v heat-
inactivated fetal bovine serum (FBS) and supplemented with 100 IU/ml penicillin-100 μg/ml
streptomycin solution and 2 mM L- glutamine. Cells were then seeded in a clear 96-well plate at an
density of 5 x 104 cells/well in 200μl of complete cell culture medium. After 24h, cells are exposed to
the chosen nanoparticle (Copper Oxide) and incubated for 24h. The untreated cells were then
incubated with 0.1% v/v Triton X-100 (TX-100) cell death control or 2 mM* carbonyl cyanide- 4-
(trifluoromethoxy)phenylhydrazone (FCCP) mitochondrial activity control for 15 min.

Fluorescence staining and imaging

For cell health and morphology assessment, cells were stained using a dye cocktail containing
Hoechst 33342 (nuclear stain) 20 μg/mL, MitoTracker Red (mitochondrial stain) 600 nM and Image-It
Dead Green (viability stain for cell membrane permeability) 50 nM for 30 minutes at 37 oC and then
fixation using paraformaldehyde (4%). Cells are stained with Cell Mask Deep Red (cell delineation
stain) overnight. The stained cells were washed once with PBS before imaging.

Quantitative high content analysis

An Invitrogen’s EVOS M7000 Imaging System with fluorescence, image stitching and the
accompanying Celleste Image Analysis Software was used to capture and analyse the H441 cell line
exposed to several concentrations of nanoparticle. The fluorescently labelled cells were segmented to
a cell-by-cell bases with the use of the Hoechst nuclear and Cell Mask stains. These are used to
identify the nucleated cells and highlight the cytoplasmic regions of those cells respectively. Several
parameters can then be examined including MitoTracker Red which detects changes in the
mitochondrial membrane potential and Image-It Green Dead which identifying compromised plasma
membrane of cells.

Results

The initial observations seen from high content imaging showed a dose dependent toxicity with cells
treated with nanoparticles (Copper Oxide, Figure-1). As the nanoparticle concentration is decreased
the number of cells is seen to increase indicating a reduction in cell death.
Drug Delivery to the Lungs, Volume 33, 2022 – Altin Kocinaj et al

Figure 1: Effect of Copper Oxide on the H441 population at different concentrations of nanoparticle

The method developed by Hoffman et. al.(7) allows for the development of distributions for the
parameters of interest extracted from the fluorescent images through HCA and Celleste. By comparing
the different concentration exposures of nanoparticles, they show discernible changes against each
other and untreated cells. The distributions of several parameters of a Copper oxide nanoparticle are
displayed in Figure-2. The distribution for cell area, show a reduction in the size of the cell in relation to
increasing dose of nanoparticle. At the maximum exposed dose of 100µg/ml no distribution is present
due to there being no cells visible for segmentation and quantification, confirmed by the respective
dose in the image in Figure-1.

Membrane permeability is shown to be affected by differing nanoparticle concentration, the 33µg/ml

concentration is seen to sizable number of cells (~20% of cells) are between membrane permeability
intensity 2 and 3 which is at around 5% in the lowest concentration of 0.4µg/ml. This suggests that the
nanoparticle exposure at the highest concentration causes a larger level of membrane permeability in
the cell population in comparison to the lowest concentration. This is somewhat inversely correlated to
the Mitochondrial activity, which is seen by the initial peak at the highest concentrations 33 and
11µg/ml (part of the population having low mitochondrial activity) whereas the lower concentrations
and control have a number of cells with high mitochondrial activity.

Frequency

Figure 2: Effect of different concentrations of Copper Oxide nanoparticles on selected parameters

which include Cell Area, mean diameter, membrane permeability and mitochondrial activity, against
the frequency. * 100µg/ml concentration not shown due to the lack of cells available for quantification.

Conclusions

These preliminary results show the ability of HCA to segment cells and allow for quantification of the
minute morphometric changes produced by nanoparticle toxicity. The information obtained by cell-by-
cell quantification will allow for the analysis of high-density data for different nanoparticle concentration
exposed cells with a higher degree of fidelity then a generalised one value absorbance reading for an
entire population from more standard assays. The use of statistical descriptors as well as skewness
and kurtosis will be used to describe these distributions and compare the distributions between other
nanoparticles or repeats. The use of statistical tests such as Kolmogorov Smirnov (which quantifies
distance between two distributions) will also be used to compare distributions and give statistical
significance where possible. However, this will require further enquiry into the analysis of the
distributions and the addition of standard viability assays, protein, and gene expression studies to build
an understanding and complete picture of the mechanism of action of these toxic agents. Regarding
the distributions a method of normalisation may be established such as Z-score normalisation for
comparison with other plates. Additionally, dimensionality reduction (such as Principal Component
Analysis) could also be used to correlate similar features such as cell area and diameter to reduce the
number of features, saving on computation power and developing a clearer view of the nanoparticle’s
toxicity profile(8). This in all looks at bridging the gap between cell culture and animal models with the
aim to follow the principles of the 3Rs and develop a standalone model to be used for both toxicology
and drug delivery testing purposes.

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