Plate reader spectroscopy as an alternative to atomic absorption spectroscopy for the assessment of nanoparticle cellular uptake

Fundamental studies investigating the biological effects induced by nanoparticles (NPs) explicitly require the correct assessment of their intracellular concentration. Ultrasensitive atomic absorption spectroscopy (AAS) is perceived as one of the gold standard methods for quantifying internalized NPs. Besides its limitation to metal-based NPs though, AAS also requires specific infrastructure and tedious sample preparation and handling, making it time-consuming and cost-intensive. Herein we report on a reliable, rapid, and affordable alternative to AAS – plate reader spectroscopy (PRS), which offers an accessible option for everyday laboratory use without sophisticated instrumentation. Our results demonstrate, that following a proper methodological approach, data on intracellular concentration of NPs obtained by PRS are fully comparable to AAS results. Specifically, the intracellular concentration of magnetite NPs coated with sodium oleate and bovine serum albumin in human alveolar A549 cells was assessed by PRS and AAS in parallel, with a remarkable correlation coefficient of R = 0.9914.


Introduction
The growing interest in the use of nanoparticles (NPs) for medical purposes is constantly increasing the demand for simple methods enabling their reliable intracellular quantification. Knowledge of the intracellular concentration of NPs is especially important as most of the biological effects manifest in a dose-dependent manner [1,2,3,4,5,6].
Since there is no commonly accepted approach for studying NPs in a biological environment, it is hardly possible to derive generalizable conclusions from different studies. A plethora of analytical methods is employed to measure the cellular uptake of NPs. While microscopy methods dominate in qualitative evaluation, spectroscopy methods including atomic absorption spectroscopy (AAS) [7,8], inductively coupled plasma-mass spectrometry [9,10,11], inductively coupled plasma-atomic emission spectroscopy [12], flow cytometry or confocal imaging [2,13,14,15] are preferred for quantitative assessment.
AAS is a destructive method commonly used for qualitative and quantitative analysis of metallic elements in the samples [16,17,18]. It enables ultrasensitive detection of elements across a wide range of concentrations with excellent repeatability, requires as short as 10-15 s for a single measurement, and offers a possibility for automatization [19]. On the other hand, AAS requires expensive instrumentation with skilled operators, and its use is often limited due to the time-consuming sample preparation as well as the requirement for relatively large volumes of samples (0.5-5.0 mL), which makes it impractical, especially for screening studies [2,16].
Regardless of all the indisputable benefits offered by AAS, these drawbacks open up space for alternative analytical methods with adequate sensitivity that are more suitable for daily laboratory practice.
One of the rapid and affordable methods enabling the quantification of substances is plate reader spectroscopy (PRS), suitable for absorbance measurements in the UV-VIS spectrum. Plate readers are accessible in most laboratories and are widely used for screening experiments as they allow for measuring multiple samples at once [20].
Herein we investigated the suitability of PRS for routine quantification of intracellular concentration of NPs based on their absorbance, as an accessible alternative to AAS. Within this study, bovine serum albumin (BSA) and sodium oleate (SO) coated magnetite NPs (BSA-SO-MNPs) were used, as they are biocompatible and have favorable toxicity profiles [21,22,23,24]. The metallic core enabled parallel quantification of NPs by AAS. Since lungs are well-known to accumulate iron oxide NPs [25,26], intracellular uptake of BSA-SO-MNPs was evaluated in human alveolar epithelial cancer cell line A549.
Cells were cultured in a humidified atmosphere with 5% CO 2 at 37 C.

Magnetite nanoparticles
In this study, spherical NPs with Fe 3 O 4 core coated with SO and BSA (Table S1) were used. BSA-SO-MNPs were prepared, characterized, and kindly provided by Vlasta Zavisova, Ph.D., Institute of Experimental Physics, SAS, Kosice, Slovakia [27].

Exposure of cells to BSA-SO-MNPs
For PRS measurements, cells were seeded onto 6-well plates (Ø ¼ 35 mm) at a density of 2 Â 10 5 cells/well. After 24 h, cells were pre-treated in a culture medium with 2% FCS for 24 h. Cells were then exposed to various non-toxic concentrations [25,26]  For AAS analysis, cells were seeded on Petri dishes (Ø ¼ 100 mm) at the density of 1 Â 10 6 cells/dish. After 24 h, cells were pre-treated in the medium with 2% FCS for 24 h. Cells were then exposed to various non-toxic concentrations [25,26] of BSA-SO-MNPs ranging between 0-116 μg Fe 3 O 4 /mL for 5 h in the culture medium with 2% FCS. After exposure, cells were washed with PBS, trypsinized, counted, pelleted (200 x g, 5 min), and digested using 500 μL of 65% HNO 3 (Centralchem, Slovakia) in an ultrasonic bath at 85 C for 2 h. The digests were then diluted in 2% HNO 3 solution.

UV-VIS spectroscopy
The representative UV-VIS absorption spectrum of BSA-SO-MNPs was assessed using the Shimadzu UV-1650 PC UV-VIS spectrometer (Shimadzu Corporation, Japan) for the concentration of 35.5 μg Fe 3 O 4 per mL.
The absorption maximum of BSA-SO-MNPs was 273 nm. To avoid cross-interference with the intracellular protein fraction (~280 nm), a wavelength of 350 nm was arbitrarily chosen to determine the calibration curve. In case of transmittance of a sample was below 10%, it was diluted and re-measured. The analysis was carried out using Varian AA240FS (Varian Inc., USA) atomic absorption spectrometer, equipped with a flame burner. AAS was adapted to quantify the amount of Fe in the samples with the following setting: wavelength 248.3 nm, slit width 0.2 nm, flame type: acetylene-air, flow: 2.0 L/min for acetylene, and 13.5 L/min for air, deuterium background correction, method of the calibration curve in the range 0.1-10 mg/L. The limit of detection (LOD) and limit of quantification (LOQ) for the AAS instrument were 0.002 mg/L and 0.005 mg/L, respectively. LOD and LOQ for the AAS method were 0.007 mg/L and 0.025 mg/L, respectively. All samples were analyzed in triplicate.

Plate reader spectroscopy
The amount of Fe was corrected to the signal from un-exposed control cells and used to calculate the amount of magnetite expressed as pg Fe 3 O 4 per cell.

Assessment of the absorption spectrum of BSA-SO-MNPs by UV-VIS spectroscopy
To quantify NPs in samples using PRS, it was first necessary to determine the absorption spectrum of BSA-SO-MNPs and to determine the appropriate wavelength for a reliable assessment of their absorbance. Figure 1 shows the characteristic absorption spectrum of BSA-SO-MNPs in water in the wavelength range 265-765 nm with an absorption maximum at 273 nm.
Concerning the absorption maximum of nucleic acids and proteins between 260-280 nm, the wavelength of 350 nm was arbitrarily selected for quantitative analyses, as it falls outside the cross-interference region, while still providing meaningfully high absorbance (64% of the absorption maximum) for reliable measurement of BSA-SO-MNPs concentration in the samples.
The suitability of wavelength 350 nm to determine of BSA-SO-MNPs concentration in samples was evaluated through the linearity of absorbance versus NPs concentration in terms of the Beer-Lambert law. Since linear regression of the obtained scatter had a coefficient of determination R 2 ¼ 0.9998 (Figure 1, right), wavelength 350 nm proved to be suitable for measuring the absorbance of NPs and calculating their concentration. Based on this finding, the intracellular concentration of BSA-SO-MNPs was calculated based on the absorbance assessed by PRS at 350 nm. Determination of calibration curves for BSA-SO-MNPs in different solutions by PRS was also determined ( Figure S1).

Comparison of PRS and AAS in the quantification of intracellular concentration of BSA-SO-MNPs
To assess PRS as a suitable alternative method to AAS, data on the intracellular concentration of BSA-SO-MNPs in exposed A549 cells obtained by PRS and AAS were compared head-to-head. Figure 2 shows the intracellular concentration of BSA-SO-MNPs in A549 cells determined by PRS and AAS for different applied NPs concentrations. Data showed an excellent correlation of results from both methods (Figure 3) with a coefficient of correlation R ¼ 0.9914. Moreover, the overlap of theoretical and experimental linear regressions indicates, that the absolute values obtained by PRS fully correspond with those obtained by the reference AAS method.

Discussion
Our study revealed an excellent correlation between data from AAS and PRS regarding the assessment of the intracellular concentration of NPs. Unlike AAS which measures the absorbance of Fe, PRS measures the absorbance of the entire nanoparticle, including its coating. As long as the PRS measurement results are corrected for untreated cells, the results are not biased by endogenous Fe. In addition, compared to AAS, PRS is more flexible in terms of the measured analyte, as it does not require a metal core and possibly enables the quantification of NPs based on the absorbance of the coating alone. In the latter case, it is necessary to ensure that the coating used is stable in the biological environment. For daily routine practice, as far as the analyzed NPs absorb light in the UV-VIS spectrum and it is possible to select a wavelength that does not interfere with other components in the measured samples, PRS could be used as fast cost-effective full-fledged alternative to AAS for determination of intracellular uptake of NPs.

Declarations
Author contribution statement Barbora Svitkova, Michal Selc, Veronika Nemethova, Filip Razga: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Monika Ursinyova: Performed the experiments. Andrea Babelova: Conceived and designed the experiments; Wrote the paper.

Data availability statement
Data will be made available on request.

Declaration of interests statement
The authors declare no conflict of interest.

Additional information
Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2022.e11595.