A quantitative and non-invasive method for nanoparticle translocation and toxicity evaluation in a human airway barrier model

Human exposure to environmental nanoparticles (NPs) may result in systemic distribution and accumulation of NPs. Depending on exposure conditions and their physiochemical properties, NPs could cross biological barriers and reach vital organs. This method describes an analytical technique that quantifies the nanoparticles’ translocation through a sample human airway barrier. Silver nanoparticles (AgNPs) were used as the example nanoparticles due to their common use in nanotechnology. The analytical method introduced in this study allows mass measurements of both cellular uptake and translocation of AgNPs through the modeled barrier. Additionally, cytotoxicity was evaluated using a convenient assay to investigate adverse effects from AgNPs treatment. The assay measures cellular injury from each layer in the barrier independently. The assay does not engage cells physically for chemical reaction, therefore it is non-destructive to the model, and the model can be used for other purposes subsequently. To conclude, this study provides researchers with measurable tools for evaluating the translocation, cellular trafficking, uptake and toxic effects of metallic nanoparticles in the in vitro barrier format.• Quantitative evaluation of nanoparticles translocation through human airway barrier• Non-invasive and quantifiable toxicity evaluation for co-culture models


Specifications
In vitro alternative method, nanotoxicology Method name: Quantitative evaluation of nanoparticles translocation; non-invasive toxicity evaluation in co-culture models Name and reference of original method 1 Zhang, F., et al. (2015). "Particle uptake efficiency is significantly affected by type of capping agent and cell line." Journal of Applied Toxicology 35 (10): 1114-1121.

Resource availability
Materials and supplies list (Excel file)

Method details
Protocol 1 Cell lines and culture media specification/preparation 1.1 Obtain the cell lines from a reliable source. The model uses the following cell types: human vascular endothelial cells EA.hy926, human bronchial epithelial cells Calu-3 and human acute monocytic leukemia Thp-1. Maintain the cultures following provider's instruction in proper media under appropriate culture conditions until the desired confluence. Standard culture conditions for above mentioned cells are 37 °C, humid air mixture containing 5% CO 2 . 1.2 The three selected cell types in the co-culture model each require its own type of media.
Prepare and store all media in sterile conditions, and use them in biological safety cabinets only. Product details (such as vendor and catalog number) are summarized in the material list spreadsheet in the supporting information. Take three TEER measurements for each sample to get a daily average, and compare it to 10 0 0 cm 2 to determine if additional culturing is needed. When measuring TEER in the culture, it is imperative to act efficiently to reduce artifact, as temperature change affects medium conductivity and thus the resistance reading. 3 Cellular uptake and translocation of NPs 3.1 In this study, the example NPs are 50 nm (transmission electron microscopy diameter) tanniccoated AgNPs. They have been fully characterized by the manufacturer and in house using several instrumentations on size, shape and surface charge. For detailed characterization of these nanoparticles, please refer to [1] . The cellular uptake and translocation of AgNPs were evaluated in a 24 hr exposure scenario at 37 °C and 3 mg/L dosing concentration ( Fig. 4 ). 3.1.1 Obtain or synthesize AgNPs in the pure and concentrated sterile form. The AgNPs used in the study are highly concentrated at 1 mg/ml, sterile, endotoxin-and residual reactantsfree. Once triple-culture is ready (TEER is restored to 10 0 0 cm 2 ), add 1.5 μL of the stock AgNPs to the apical side of the inserts (0.5 mL of mixed media of RPMI and DMEM-low) to final concentration of 3 mg/L. 3.1.2 Return culture to incubator and maintain at 37 °C for 24 hr. 3.2 Collect medium by aspirating from both chambers, and cells grown on apical and basal sides of the inserts. Analyze silver content in each of the following section within the co-culture. Remember to include 10 mg/L Rhodium to all calibration standard solutions. Prepare all calibration standards in 2% nitric acid with your target analyte (Ag, in this study), and at least five rising concentrations such as 0, 1, 10, 25, 50 and 100 mg/L. It is also suggested to include 2% nitric acid and calibration standard (e.g. 1 and 10 mg/L) samples in between experimental samples as quality control. 4 Cytotoxicity measurements 4.1 Before AgNPs treatment, collect conditioned medium (spent media harvested from cultured cells) from each sample to serve as control (before treatment). Media in the inset and in the receiving well need to be collected and stored separately. 4.2 Dose cells accordingly and measure cytotoxicity after 24 h. The cytotoxicity of AgNPs is evaluated by comparing lactate dehydrogenase (LDH) level in the extracellular environment (in culture medium) before and after AgNPs treatment ( Fig. 6

Additional information
Human exposure to environmental nanoparticles (NPs) becomes inevitable due to their wide range of applications in agricultural, industrial and medical fields. Depending on exposure conditions and their physiochemical properties, NPs could cross biological barriers and reach vital organs. Translocation or efflux of nanoparticles through biological barriers has been routinely evaluated by animal models, due to the architectural limitation of conventional in vitro models. New cellular models that use three-dimensional co-culture have the potential of providing more physiologically relevant condition and obtaining more predictive data. This paper introduces a method that quantitatively evaluates the translocation and toxicity of NPs in co-culture settings.
The quantification methods were adopted and improved from Zhang et al., 2015 [1] . The original method was proven valuable in the assessment of cellular uptake and toxicity in conventional monoculture models. However, the model has inherent architectural limitation and lacks the power to evaluate efflux of AgNPs in a dynamic mode. The improved coculture model, owing to the membrane insert, has an added space dimension (y axis) and is closer to a physiological barrier. Movement of NPs in both the lateral (x) and vertical (y) directions between chambers become measurable. The adapted analytical methods were applicable to use in the new model and proven useful in the quantification of mass in the dynamic setting. The quantified translocation is particularly valuable information in the assessment of drug delivery system and toxicant distribution systemically.
For the development of the coculture barrier, the protocol details the steps to assemble a sample human airway barrier model ( Fig. 1 ). The model is comprised of three cell types in a two-chamber structure including epithelial, endothelial and macrophage-like cells. The resultant barrier does not claim to be the closest mimic of human air-blood barrier, but rather serves as a template to       illustrate the steps in barrier development. The assembly method is highly adaptable. Researchers are encouraged to follow this template and customize barriers with cell types that best suit their research purposes. Additional biological barriers such as the blood-brain, intestinal, placenta barriers can also be simulated using this system. The barrier presented here was characterized for barrier integrity ( Fig. 2 ) and macrophage activation ( Fig. 3 ) in the previously published manuscript. Finally, translocation and toxicity were assessed using the method described herein, and results indicate significant efflux of AgNPs through the barrier ( Fig. 5 ) along with measurable cytotoxicity ( Fig. 7 ) [2] .
The presented barrier utilizes a physical membrane to support and separate the cells from opposite sides. One important limitation of incorporating membrane in these models is the retention of tested compound in its pores. As much as 30% of Ag was retained in the membrane as stated in our previous study (Zhang et al., 2019). The same issue was also reported by [3] where fluorescencelabelled nanobeads were absorbed on Transwell® membrane. Therefore, it is imperative to conduct an acellular retention test prior to the actual research to determine retention rate of the tested subject. Membrane inserts are available in various pore sizes. Larger pore membranes are generally more permeable, but they could also be too leaky to support cell growth. Considering features of both permeability and tissue support, we chose the 1 μm pore size for the membrane used in this study.
In this study, the authors used a Voltohmmeter to confirm barrier integrity by measuring TEER. Other methods available that inform barrier properties include immunostaining adherent and tight junction proteins (e.g. E-/VE-cadherin, ZO-1) [4] , and permeability measurements of Lucifer Yellow CH dipotassium salt transported from apical to basal side of the barrier [5] . The LDH bioassay may also be substituted by TEER measurement directly in the barrier to indicate toxicity. While a compromised membrane can result in decreased TEER [6] , the instrument may not be sensitive enough to detect minor cellular damage. Other viability screening assays, such as tetrazolium-based colorimetric assay (MTT), can be an alternative to LDH measurements, since recently there have been concerns that some metal particles could interfere with LDH reagents and produce false results [7] . However, assays like MTT and other colorimetric or fluorometric assays share one common limitation. These assays interact with cells directly by engaging cells and deprive them in the chemical reaction. The advantages of using LDH assay are noteworthy. It measures analyte in the extracellular matrix and not in cells, so the tissue remains undisturbed and can be reused for other purposes (such as uptake analysis). The reusability of costly coculture models is a valuable feature for either the in-house-made or commercial units.
LDH leakage from treatment groups are compared to the level of spontaneous leakage from the control. This is a relative comparison, considering the control as the baseline. The exact LDH concentration in the extracellular matrix of LDH can be determined by fitting the LDH data onto a pre-established LDH standard curve. Another alternative is to present LDH leakage in the% max leakage. The max LDH leakage is determined by lysing the same amount of cells with 1% of Triton X solution. The% max leakage is then calculated using the sample absorbances in the following formula: LDH (observed) / max LDH (cell lysis) * 100%.