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. 2020 Jun 19;21(12):4388.
doi: 10.3390/ijms21124388.

ESR Method in Monitoring of Nanoparticle Endocytosis in Cancer Cells

Ryszard Krzyminiewski  1 , Bernadeta Dobosz  1 , Bart Krist  2 , Grzegorz Schroeder  3 , Joanna Kurczewska  3 , Hans A R Bluyssen  2
Affiliations

ESR Method in Monitoring of Nanoparticle Endocytosis in Cancer Cells

Ryszard Krzyminiewski et al. Int J Mol Sci. .

Abstract

Magnetic nanoparticles are extensively studied for their use in diagnostics and medical therapy. The behavior of nanoparticles after adding them to cell culture is an essential factor (i.e., whether they attach to a cell membrane or penetrate the membrane and enter into the cell). The present studies aimed to demonstrate the application of electron spin resonance (ESR) as a suitable technique for monitoring of nanoparticles entering into cells during the endocytosis process. The model nanoparticles were composed of magnetite iron (II, III) oxide core functionalized with organic unit containing nitroxide radical 4-hydroxy-TEMPO (TEMPOL). The research studies included breast cancer cells, as well as model yeast and human microvascular endothelial cells. The results confirmed that the ESR method is suitable for studying the endocytosis process of nanoparticles in the selected cells. It also allows for direct monitoring of radical cellular processes.

Keywords: TEMPOL spin label; breast cancer cells; electron spin resonance; magnetic nanoparticles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Functionalized nanoparticles do not exert cytotoxicity and enter the cells. (A–C): HMEC morphology is not changed by the addition of nanoparticles without (B) or with magnet (C), compared to nontreated control cells (A). Bright field images; magnification = 10x. (D–E): Cell viability as percentage of control for (D) nanoparticles with TEMPO spin label and (E) nanoparticles with TEMPO spin label and FITC; measured by EZ4U proliferation and cytotoxicity assay. Error bars represent mean + SD. * = p < 0.05 (one-way ANOVA). (F–G): Confocal fluorescent imaging of FITC labeled nanoparticles in HMEC cells; (F) only green and blue channels, (G) fluorescent channels + bright field. Magnification = 100x.
Figure 2
Figure 2
ESR spectrum of the Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL nanoparticles recorded in a magnetic sweep range of 650 mT at 293 K.
Figure 3
Figure 3
ESR spectra of the Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL nanoparticles: (a) aqueous solution of the nanoparticles recorded at 293 K in a magnetic field sweep range of 8 mT; (b) aqueous solution of the nanoparticles recorded at 240 K in a magnetic field sweep range of 15 mT; (c) the nanoparticles with MDA-MB-231 breast cancer cells recorded at 293 K in a magnetic field sweep range of 8 mT; (d) the nanoparticles with MDA-MB-231 breast cancer cells recorded at 240 K in a magnetic field sweep range of 15 mT; (e) the nanoparticles with HMEC recorded at 293 K in a magnetic field sweep range of 8 mT; (f) the nanoparticles with HMEC recorded at 240 K in a magnetic field sweep range of 15 mT; (g) the nanoparticles with yeast cells recorded at 293 K in a magnetic field sweep range of 8 mT; (h) the nanoparticles with yeast cells recorded at 240 K in a magnetic field sweep range of 15 mT.
Figure 3
Figure 3
ESR spectra of the Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL nanoparticles: (a) aqueous solution of the nanoparticles recorded at 293 K in a magnetic field sweep range of 8 mT; (b) aqueous solution of the nanoparticles recorded at 240 K in a magnetic field sweep range of 15 mT; (c) the nanoparticles with MDA-MB-231 breast cancer cells recorded at 293 K in a magnetic field sweep range of 8 mT; (d) the nanoparticles with MDA-MB-231 breast cancer cells recorded at 240 K in a magnetic field sweep range of 15 mT; (e) the nanoparticles with HMEC recorded at 293 K in a magnetic field sweep range of 8 mT; (f) the nanoparticles with HMEC recorded at 240 K in a magnetic field sweep range of 15 mT; (g) the nanoparticles with yeast cells recorded at 293 K in a magnetic field sweep range of 8 mT; (h) the nanoparticles with yeast cells recorded at 240 K in a magnetic field sweep range of 15 mT.
Figure 4
Figure 4
An example of changes in ESR spectra of Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL recorded at 240 K, depending on incubation time of the nanoparticle solution with yeast cells: (a) the beginning of incubation, (b) after 30 min of incubation, (c) after 90 min of incubation, (d) after 180 min of incubation.
Figure 5
Figure 5
Changes in ESR signal intensity of Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL nanoparticles with yeast cells depending on incubation time.
Figure 6
Figure 6
Microscopic photos of yeast cells in a solution with Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL nanoparticles: (a) the beginning of incubation, (b) after 3 h of incubation, and yeast cells without nanoparticles: (c) the beginning of incubation, (d) after 3 h of incubation.
Figure 7
Figure 7
Confocal microscope images showing the entry of Fe3O4@SiO2@FITC@Dextran-TEMPOL nanoparticles into yeast cells.
Figure 8
Figure 8
ESR spectra of TEMPOL attached to Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL: (a) a so-called broad experimental triplet, (b) a broad simulated triplet, (c) a narrow experimental triplet, (d) a narrow simulated triplet.
Figure 9
Figure 9
Changes in the structure and intensity of ESR spectra in the endocytosis process of the functionalized nanoparticles in a cell: A—ESR signal from the spin label not bonded to cells, B—ESR signal from the spin label attached to a cell, C—ESR signal from the spin label located inside cells (in organelles such as endosome or lysosome), D—ESR signal from the spin label probably present in cellular mitochondria. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com.
Figure 10
Figure 10
Changes in the intensity of the spin label attached to the magnetic nanoparticles during incubation with cells. The approximation was made according to Equation (3).
Figure 11
Figure 11
The recombination of TEMPOL spin label.
Figure 12
Figure 12
The structures of Fe3O4@SiO2@SiNHDOX@Dextran-TEMPOL (a) and Fe3O4@SiO2@FITC@Dextran-TEMPOL (b) nanoparticles.
See this image and copyright information in PMC

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