Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Summary

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Abstract

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Introduction

Metal-organic frameworks (MOFs) are hybrids formed through the combination of metal ions and organic linkers^1,2 in organic solvents. Due to the variety of such combinations, MOFs possess structural diversity³, tunable porosity, high thermal stability, and high surface areas^4,5. Such characteristics make them attractive candidates in a variety of applications, from gas storage^6,7 to catalysis^8,9, and from contrast agents^10,11 to drug delivery units^12,13. However, the implementation of MOFs into such applications has raised concerns relative to their safety to both the users and the environment. Preliminary studies have shown, for instance, that cellular function and growth change upon the exposure of cells to metal ions or linkers used for MOF synthesis^1,14,15. For instance, Tamames-Tabar et al. demonstrated that ZIF-8 MOF, a Zn-based MOF, was leading to more cellular changes in a human cervical cancer cell line (HeLa) and a mouse macrophage cell line (J774) relative to Zr-based and Fe-based MOFs. Such effects were presumably due to the metal component of ZIF-8 (i.e., Zn), which could potentially induce cell apoptosis upon framework disintegration and Zn ion release¹. Similarly, Gandara-Loe et al. demonstrated that HKUST-1, a Cu-based MOF, caused the highest reduction in mouse retinoblastoma cell viability when used at concentrations of 10 µg/mL or greater. This was presumably due to the Cu metal ion incorporated during the synthesis of this framework, which, once released, could induce oxidative stress in the exposed cells¹⁵.

Moreover, analysis showed that the exposure to MOFs with different physicochemical characteristics could lead to varying responses of exposed cells. For instance, Wagner et al. demonstrated that ZIF-8 and MIL-160 (an Al-based framework), used in the exposure of an immortalized human bronchial epithelial cell, led to cellular responses dependent on frameworks' physicochemical properties, namely hydrophobicity, size, and structural characteristics¹⁶. Complementarily, Chen et al. demonstrated that a concentration of 160 µg/mL MIL-100(Fe) exposed to human normal liver cells (HL-7702) caused the largest loss in cellular viability, presumably due to the metal component of this specific framework (i.e., Fe¹⁷).

While these studies categorize MOFs' deleterious effects on cellular systems based on their physicochemical characteristics and exposure concentrations, thus raising potential concerns with framework implementation, especially in biomedical fields, most of these evaluations are based on single time point colorimetric assays. For instance, it was shown that when (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) and water-soluble tetrazolium salt (WST-1) assays were used, these biochemical reagents could lead to false positives upon their interactions with the particles that the cells were also exposed to¹⁸. The tetrazolium salt and neutral red reagents were shown to possess a high adsorption or binding affinity onto the surfaces of the particles, resulting to agent signal interference¹⁹. Moreover, for other types of assays, such as flow cytometry, which was previously shown to be used for assessing changes in cells exposed to MOFs^20,21, it was shown that major issues have to be circumvented if a viable analysis of particles' deleterious effects is to be considered. In particular, detection ranges of the particles' sizes, especially in mixed populations like the ones offered by MOFs or references of the particles used for calibration before cellular changes, have to be addressed²². It was also shown that the dye used during cell labeling for such cytometry assays could also interfere with the nanoparticles that the cells were exposed to²³.

The goal of this study was to use a real-time, high-throughput evaluation assay to assess changes in cell behavior upon exposure to a select MOF. Real-time evaluations can help provide insights into time-dependent effects, as related to the windows of exposures¹⁶. Further, they provide information on changes in cell-substrate interactions, cell morphology, and cell-cell interactions, as well as how such changes depend on the physicochemical properties of the materials of interest and exposure times^24,25 respectively.

To demonstrate the validity and applicability of the proposed approach, human bronchial epithelial (BEAS-2B) cells, ZIF-8 (a hydrophobic framework of zeolitic imidazolate¹⁶), and electric cell-substrate impedance sensing (ECIS) were used. BEAS-2B cells represent a model for lung exposure²⁶ and have been previously used to evaluate changes upon the exposure of cells to nanoclays and their thermally degraded byproducts^26,27,28, as well as assess the toxicity of nanomaterials, such as single-walled carbon nanotubes (SWCNTs)¹⁸. Furthermore, such cells have been used for more than 30 years as a model for pulmonary epithelial function²⁹. ZIF-8 was chosen due to its wide implementation in catalysis³⁰ and as contrast agents³¹ for bioimaging and drug delivery³², and thus for the extended potential for lung exposure during such applications. Lastly, ECIS, the noninvasive, real-time technique, was previously used to evaluate changes in cell adherence, proliferation, motility, and morphology^16,26 as a result of a variety of interactions between analytes (both materials and drugs) and exposed cells in real-time^16,18,28. ECIS uses an alternating current (AC) to measure the impedance of cells immobilized on gold electrodes, with the impedance changes giving insights into changes in resistance and capacitance at the cell-gold substrate interface, barrier function as induced by cell-cell interactions, and over-cell layer coverage of such gold electrodes^33,34. Using ECIS allows quantitative measurements at a nanoscale resolution in a noninvasive, real-time manner^26,34.

This study assesses and compares the simplicity and ease of evaluation of MOF-induced changes in cellular behavior in real-time with single-point assay evaluations. Such a study could be further extrapolated for evaluating cell profiles in response to exposure to other particles of interest, thus allowing for safe-by-design particle testing and subsequent helping with implementation. Moreover, this study could complement genetic and cellular assays that are single-point evaluations. This could lead to a more informed analysis of the deleterious effects of particles on the cellular population and could be used for screening such particles' toxicity in a high-throughput manner^16,35,36.

Protocol

1. ZIF-8 synthesis

1. For the purpose of this example, use a 1:10:100 (metal:linker:solvent) mass ratio to synthesize the ZIF-8. For this, measure out zinc nitrate hexahydrate, and record the measurement. Utilize the example mass ratio to calculate the amount needed for the linker, 2-methylimidazole, and the solvent (i.e., methanol).
2. Place the zinc nitrate hexahydrate and linker into two different glass vials. Add half of the calculated amount of methanol to the zinc nitrate hexahydrate and the other half to the linker. Allow each solution to dissolve.
3. Combine the two solutions (i.e., solutions containing the metal and linker, respectively). Place the container with the combined solution for the reaction onto a stir plate and stir at 700 rpm for 24 h at room temperature (RT).

2. ZIF-8 collection

1. Once the above reaction has concluded (i.e., after 24 h), place the solution into 50 mL centrifuge tubes and centrifuge at 3,075 x g for 5 min (use equal amounts and balance the rotor). Remove any of the supernatants from the centrifuged tubes.
2. Add methanol (5-15 mL) to the centrifuge tubes and redisperse the particles.
3. Repeat the centrifugation and washing steps at least two to four more times. After the last wash, discharge the supernatant.
   NOTE: The washing steps remove any non-precipitated species.
4. Remove the lids of the tubes and place parafilm tape over the top; subsequently, poke holes in the parafilm. Place the tube/s containing the sample in a vacuum chamber at RT to dry for 24 h.

3. ZIF-8 surface morphology (scanning electron microscopy [SEM])

1. Mount the dry powders of ZIF-8 onto carbon tape and sputter for 120 s with gold-palladium to prevent any charging that could occur during the SEM analysis.
2. Set the accelerating voltage of the microscope to 15 kV. Bring the particles into focus and adjust the magnification (magnifications of 500x, 1,000x, 1,500x, 4,000x, 10,000x, 20,000x, 30,000x, and 40,000x were used.)
3. Image the particles under the magnification that allows both a clear particle size and morphology assessment (recommended to consider ranges for particles of 5 µm, 10 µm, and 20 µm).
4. Collect data from at least three different fields of view, and for each, consider different magnifications.

4. ZIF-8 elemental composition

1. Follow the steps for SEM imaging.
2. Using the energy-dispersive X-ray (EDX) spectroscopy unit of the SEM microscope, analyze the sample elemental composition.
3. Make sure the SEM accelerating voltage and magnification match the EDX monitor. Turn off the infrared camera on the SEM microscope.
4. On the EDX monitor, go to Collect Spectrum and input a collection time of 200 s. This is recommended to avoid particle charging and disintegration that could lead to false evaluations.
5. Collect data from at least three different fields of view. Once the collection is finished, analyze the data, and identify the elements of interest.

5. Cell culture

1. Culture immortalized BEAS-2B cells in media supplemented with 5% fetal bovine serum (FBS) and 0.2% penicillin/streptomycin.
   NOTE: Passages between 5-10 should be used to ensure that no phenotypical changes have previously taken place in the cell batch being used³⁷ (all reagents are listed in the Table of Materials).
2. Take a 100 mm cell culture plate and place 10 mL of the media onto the plate. Add 1 mL of the BEAS-2B cell solution to the plate.
3. Place the plate into an incubator (37 °C, 5% CO₂) and allow the cells to grow.
4. Check the plate after 24 h to determine if the cells have reached 80% confluency. Herein, the 80% confluency refers to the percentage of the surface covered by adhered cells. If not, change the media and allow the cells to grow until the 80% confluency level is reached.

6. Cell counting

1. Once the cells on the plate have reached a confluency of 80%, remove the media from the plate. Wash the plate with 5 mL of phosphate-buffered saline (PBS).
2. Remove the PBS from the plate. Add 2 mL of trypsin to the plate and place it in the incubator (37 °C, 5% CO₂) for 2 min.
3. Add 2 mL of media to the plate and pipet the media up and down to remove the cells from the plate. Transfer the solution to a 15 mL tube and centrifuge at 123 x g for 5 min.
4. Remove the supernatant from the tube and add 2 mL of media. Pipet the media up and down to redisperse the cells.
5. Using a 1.5 mL tube, add 20 µL of trypan blue and then 20 µL of the cell solution (1:1 ratio of trypan blue:cell solution).
6. Place 10 µL of the cell mixture onto a glass slide and place it onto an automatic cell counter. Wait until the screen comes into focus, then select capture.
7. The screen displays the live cell numbers and cell viability. The measurement range extends from 1 x 10⁴-1 x 10⁷ cells.
8. Alternatively, count the cells manually using a hemocytometer.
   1. For this, add 15-20 µL of the trypan blue-treated cell solution to the hemocytometer.
   2. Use an upright microscope with a 10x objective focus on the grid lines of the hemocytometer.
   3. Count the number of cells in the four outer squares and divide the number by four. Then, multiply by 10,000 to obtain the live cell count number.
   4. To calculate cell viability, add together the live and dead cell count to obtain the total cell count, and subsequently divide the live cell count by the total cell count.

7. ZIF-8 dose preparation

1. Make a ZIF-8 stock solution of 1 mg/mL. For this, measure the amount of ZIF-8 and add deionized (DI) water to the particles to reach a 1 mg/mL concentration.
2. Sonicate (set the sonicator to sonics) the ZIF-8 stock solution for 2 min at 30 s intervals in a water bath.
3. Make different doses of ZIF-8 for cellular exposures by calculating the amount of stock solution and Dulbecco's modified eagle medium (DMEM) needed to reach the desired doses using the equation C₁V₁ = C₂V₂. The doses will range from 0-950 µg/mL.
   NOTE: In this experiment, doses of 0 µg/mL, 1 µg/mL, 10 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, 500 µg/mL, 750 µg/mL, and 950 µg/mL were selected.

8. Half-maximal inhibitory concentration (IC  ₅₀)

1. After cell counting, seed the BEAS-2B cells at a density of 4 x 10⁴ cells/mL in a 96-well plate, with a volume of 100 µL/well.
   1. To reach a density of 4 x 10⁴ cells/mL, use C₁V₁ = C₂V₂ to calculate the amount of cell solution needed to be combined with the media.
   2. Ensure to maintain blank wells for each dose; leave these wells empty until ZIF-8 exposure.
2. Place the 96-well plate into the incubator at 37 °C and 5% CO₂ and allow the cells to grow to a confluent monolayer for 24 h.
3. Remove the media from the wells and expose the BEAS-2B cells to ZIF-8 doses ranging from 0-950 µg/mL. Add 100 µL of ZIF-8 to their respective blank and cell wells.
4. Place the 96-well plates back into the incubator for an exposure time of 48 h.
5. After the exposure, add 10 µL of 4-[3-(4-idophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) to each well (blank and cell wells) and incubate for 2 h. The media remains in the wells; the ratio is 1:10 (reagent:media).
6. Read the changes in absorbance on a plate reader using a 485 nm absorbance.
7. Calculate the cell viability through the software to determine the IC₅₀ value (see section 10).

9. Electrical cell-substrate impedance sensing (ECIS)

1. Use a 96-well plate (96W10idf) to perform ECIS analysis. The plate²⁸ contains inter-digitated finger connection electrodes covering an area of about 4 mm².
2. First, test that all the wells are being read by the sensors. For this, place the 96-well plate onto the well station and click on the Setup button.
3. If the wells are connected properly, green will appear; any unconnected wells will be identified as red. If such a case arises, remove and reinsert the well plate and click the Setup button again, until all the wells offer viable green readings.
4. Stabilize the electrodes for 40 min with 200 µL of media per well to prevent potential drift. Add 200 µL of media to each well being used and then place the 96-well plate onto the ECIS station. Click on Setup/Check to ensure the plate is properly connected. Click on Stabilize.
5. Once stabilization is complete, remove the plate from the station and remove the media from each well.
6. Seed the BEAS-2B cells at a density of 4 x 10⁴ cells/mL, with a volume of 100 µL per well, into the wells containing the cells and place the plate onto the ECIS station in the incubator.
7. On the monitor, click on Check to determine that all the electrodes are read by the sensors on the station.
8. Set up the time course measurement by selecting Multiple Freq/Time (MFT). The program will measure each well at seven different frequencies.
9. Click Start and allow it to run for 24 h so the cells can form a confluent monolayer.
10. After 24 h, the wells containing cells will show increased resistance on the monitor. This is an indicator that cells have attached to the electrodes.
11. Press Pause on the monitor to stop the data collection.
12. Make the ZIF-8 doses at, above, and below the calculated IC₅₀ value by following the steps outlined in section 7 above.
13. Expose the ZIF-8 nanoparticles to their respective blank and cell wells at a volume of 100 µL per well and place the 96-well plate back onto the station.
14. Click on Check again on the monitor to ensure the sensors are reading all the electrodes. Click on Resume and allow the system to run for 72 h to monitor the cellular behavior and attachment.
15. Once data collection has finished, click on Finish on the monitor. To save the file, click File > Save As. Save the data as a spreadsheet file.
16. To get the alpha parameters, click on Model. On the screen pop-up, click on Media-Only Well, then select the well that has only media.
17. Click on Set Ref. On the monitor, click on Find.
18. If the well shown in the system is not the same as what was chosen, repeat step 9.16.
19. Click on Set. Click on Fit T-Range to determine the time range desired for data collection.
20. While it is processing, click on Alpha. When the system finishes the analysis, click on Save RbA and save it as a spreadsheet file.
    NOTE: Refer to the ECIS manual, which can be found on the manufacturer's website³⁸.

10. Data analysis

1. SEM
   1. Open the imaging software to analyze the SEM images. Click File > Open, and then select the image to analyze.
   2. Click on Image > Type > 8 Bits to convert the image to grayscale to perform the threshold operation.
   3. Calibrate the distance measurement of pixels to microns. Click on the Straight-Line tool and trace the length of each particle aimed to be measured.
   4. Click Analyze and then Set Scale. The known distance will be set to the length of the size of the scale bar on the SEM image. The known unit of length will be set to microns. Check Global and press Ok.
   5. Click on the Straight-Line tool and trace the diameter of the particle, then select Analyze and Measure. Record the length result.
   6. Repeat for all the collected images. Average all the diameters from at least 60 particles for a suitable statistics evaluation.
2. EDX
   1. Average all the weight percentages of each element together from all the collected images (see EDX section above).
   2. Using a spreadsheet, plot a bar graph of each element on the x-axis and their average elemental weight percentage on the y-axis.
3. IC₅₀​
   1. Average the blank wells and average the dose wells for each replicate.
   2. Calculate the adjusted blank by subtracting the control blank average from the dose blank average. Calculate the true cell value for each dose by subtracting the adjusted blank from the average dose value.
   3. Subtract the adjusted blank value for each dose from the average control value. Calculate the cell viability of each dose utilizing the equation: cell viability = (true cell value/(control value - adjusted blank value)) x 100.
   4. Repeat this for all other replicates.
   5. Average the cell viability for each replicate together to get the final cell viability for each dose.
   6. In the software, choose the XY tab on the screen.
   7. Enter the concentration (dose) values into the X column and the cell viability (%) from each dose into the Y column.
   8. Transform the X values by clicking Analyze > Transform > Ok, and Transform X = Log(X).
   9. Normalize the Y values by selecting the Transformed Data sheet, clicking on Analyze, and then selecting Normalize.
   10. Select the Normalize of Transform data sheet, click Analyze, and select Nonlinear Regression (Curve Fit). Select Dose-Response-Inhibition and click Log(Inhibitor) Vs. Response-Variable Slope (Four Parameters). Click on OK to view the results.
4. ECIS
   1. From the spreadsheet, take the resistance (ohm) columns for each replicate (blank wells and dose wells) and average them together from the 4,000 Hz frequency. This frequency allows cell-cell contact evaluation³⁸.
   2. Calculate the corrected resistance by subtracting the averaged blank from the averaged dose for each replicate. Average the corrected resistance for the replicates for each dose.
   3. Repeat the same steps for the alpha values to calculate the average alpha parameter. Plot the x-axis as time (h) and the y-axis as the average resistance/alpha values for each dose value.

11. Statistical analysis

1. SEM
   1. Perform a standard deviation in the spreadsheet file where the average diameters of the ZIF-8 particles were calculated.
   2. Utilizing the STDEV function in the spreadsheet, highlight the data of the 60 particles and click Enter. Graph the standard deviation for each average diameter calculated.
2. EDX
   1. Similar to SEM (steps 11.1.1-11.1.2), calculate the standard deviation for each average elemental composition using the STDEV function in the spreadsheet. Graph the standard deviations for each average elemental composition.
3. IC₅₀​
   1. Open the software utilized to calculate the IC₅₀ and click on Grouped Data Sheet.
   2. Enter the ZIF-8 doses in the rows labeled Group A, Group B, etc. Enter the average cell viability for each dose and replicate in the column corresponding with the ZIF-8 dose.
   3. Click on Analyze, and under Column Analyses, select One-way ANOVA (and nonparametric or mixed).
   4. In the Experimental Design tab, select No Matching Or Pairing. Under Gaussian Distribution Of Residuals, select Yes use ANOVA. Finally, select Yes Use Ordinary ANOVA Test under Assume Equal SDs.
   5. Under the Multiple Comparisons tab, select Compare the Mean of Each Column with the Mean of a Control Column. Click on OK to view the results.

Results

Using a common in vitro model cell line³⁹ (BEAS-2B), this study aimed to demonstrate the feasibility and applicability of ECIS to assess changes in cell behavior upon exposure to a lab-synthesized MOF. These changes assessment was complemented by analysis through conventional colorimetric assays.

The physicochemical characteristics of the framework were first evaluated to ensure the reproducibility of the methods employed, the validity of the obtained...

Discussion

Previous analysis showed that ECIS could be used to assess the behavior of cells exposed to analytes (i.e., carbon nanotubes³⁵, drugs⁴³, or nanoclays¹⁶). Furthermore, Stueckle et al. used ECIS to evaluate the toxicity of BEAS-2B cells exposed to nanoclays and their byproducts and found that the cellular behavior and attachment were dependent on the physicochemical characteristics of such materials⁴². Herein, we proposed to det...

Materials

Name	Company	Catalog Number	Comments
4-[3-(4-idophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1 assay)	Roche	5015944001
0.25% Trypsin-EDTA (1x)	Gibco	25255-056
100 mm plates	Corning	430167
1300 Series A2 biofume hood	Thermo Scientific	323TS
2510 Branson bath sonicator	Process Equipment & Supply, Inc.	251OR-DTH
2-methylimidazole, 97%	Alfa Aesar	693-98-1
5 mL sterile microtube	Argos Technologies	T2076S-CA
50 mL  tubes	Falcon	352098
96W10idf well plates	Applied Biophysics	96W10idf PET
96-well plates	Fisherbrand	FB012931
Biorender	Biorender	N/A
Countess cell counting chamber slides	Invitrogen	C10283
Countess II FL automated cell counter	Life Technologies	C0916-186A-0303
Denton Desk V sputter and carbon coater	Denton Vacuum	N/A
Dimethly sulfoxide	Corning	25-950-CQC
DPBS/Modified	Cytiva	SH30028.02
Dulbecco's modified Eagle medium	Corning	10-014-CV
ECIS-ZΘ	Applied Biophysics	ABP 1129
Excel	Microsoft	Version 2301
Falcon tubes (15 mL)	Corning	352196
Fetal bovine serum	Gibco	16140-071
FLUOstar OPTIMA plate reader	BMG LABTECH	413-2132
GraphPad Prism Software (9.0.0)	GraphPad Software, LLC	Version 9.0.0
HERAcell 150i CO2 Incubator	Thermo Scientific	50116047
Hitachi S-4700 Field emission scanning electron microscope equipped with energy dispersive X-ray	Hitachi High-Technologies Corporation	S4700 and EDAX TEAM analysis software
ImageJ software	National Institutes of Health	N/A
Immortalized human bronchial epithelial cells	American Type Culture Collection	CRL-9609
Isotemp freezer	Fisher Scientific
Methanol, 99%	Fisher Chemical	67-56-1
Parafilm sealing film	The Lab Depot	HS234526A
Penicillin/Steptomycin	Gibco	15140-122
Sorvall Legend X1R Centrifuge	Thermo Scientific	75004220
Sorvall T 6000B	DU PONT	T6000B
Trypan blue, 0.4% solution in PBS	MP Biomedicals, LLC	1691049
Vacuum Chamber	Belart	999320237
Zinc Nitrate Hexahydrate, 98% extra pure	Acros Organic	101-96-18-9