Determination of the limits for quantification of the degree of internalization of γ-Fe2O3 nanoparticles by cultures of human mesenchymal stromal cells

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Abstract

A culture of human bone marrow mesenchymal stromal cells (MSCs) was investigated in the present work. Cell culture was grown as a monolayer in a nutrient medium into which a stabilized aqueous suspension of magnetic nanoparticles (MNPs) of maghemite (γ-Fe2O3) were added. MNPs were synthesized by the electrophysical method of laser target evaporation. A method has been proposed for stabilizing a suspension in a nutrient medium with high ionic strength. A qualitative assessment of the possibility of internalization (either by fixing on the cell membrane or by penetrating into the cell space) of MNPs with human MSCs was carried out using optical, scanning and transmission electron microscopy and SQUID magnetometry. Comparative analysis of the structure and magnetic properties was made, and assumptions about the features of MNP internalization in this system were provided. It has been established that the limiting value for MNPs that can reliably be analyzed in a biological sample of the type under consideration with nanoparticles of this type is of about 0.005 mg. It was found that in the considered range of initial concentrations of magnetic nanoparticles in biological samples based on human MSCs, the level of accumulation of magnetic nanoparticles in cell cultures depends on their concentration.

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About the authors

E. A. Burban

Уральский федеральный университет

Author for correspondence.
Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Мира, 19, Екатеринбург, 620002

F. A. Fadeyev

Институт медицинских клеточных технологий; Уральский государственный медицинский университет

Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Карла Маркса, 22А, Екатеринбург, 620026; ул. Репина, 3, Екатеринбург, 620028

A. P. Safronov

Уральский федеральный университет; Институт электрофизики Уральского отделения РАН

Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Мира, 19, Екатеринбург, 620002; ул. Амундсена, 106, Екатеринбург, 620016

F. A. Blyakhman

Уральский федеральный университет; Уральский государственный медицинский университет

Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Мира, 19, Екатеринбург, 620002; ул. Репина, 3, Екатеринбург, 620028

T. V. Terziyan

Уральский федеральный университет

Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Мира, 19, Екатеринбург, 620002

D. S. Neznakhin

Уральский федеральный университет

Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Мира, 19, Екатеринбург, 620002

A. A. Yushkov

Уральский федеральный университет

Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Мира, 19, Екатеринбург, 620002

G. V. Kurlyandskaya

Уральский федеральный университет

Email: e.a.mikhnevich@urfu.ru
Russian Federation, ул. Мира, 19, Екатеринбург, 620002

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Supplementary files

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2. Fig. 1. General view of a Petri dish with a dried sample of MSC culture at a concentration of MNPs of 256 μg/cm2 and markings by zones. Zone "2" is shown after washing off the biomaterial.

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3. Fig. 2. TEM images of MNP suspension (a) and microdiffraction pattern confirming the phase composition: γ-Fe2O3 (b). Size distribution of MNPs constructed based on TEM data for images of 1005 particles (c)

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4. Fig. 3. Distribution of particles/aggregates in a suspension of maghemite nanoparticles stabilized with sodium citrate. 1 – in water; 2 – in DMEM nutrient medium.

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5. Fig. 4. Average hydrodynamic diameter of particles/aggregates upon introduction into DMEM nutrient medium of a suspension of maghemite nanoparticles stabilized with sodium citrate and ammonium polymethacrylate using a two-stage method, depending on the time of preliminary holding of the suspension at 25°C.

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6. Fig. 5. General view of the culture of human mesenchymal stromal cells on culture plastic.

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7. Fig. 6. MSC cell culture with MNP suspension at a concentration of 1 μg/cm2 after washing (a, c), MSC cell culture with MNP suspension at a concentration of 256 μg/cm2 after washing (b, d). The upper row is in 2D, and the lower row is in 3D representation. All units on the horizontal axes are μm.

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8. Fig. 7. TEM image of a biological sample representing an ethanol wash of a MSC cell culture with a MNP suspension at a concentration of 256 μg/cm2: parts (a) and (b) show different areas where MNPs are either inside or on the surface of MSC cells. In the case of a typical area shown in photograph (c), this is an MNP agglomerate formed in the culture medium during the same study.

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9. Fig. 8. Scheme illustrating the process of interaction of MNPs added to the nutrient medium and the cell culture. (a) coagulation of MNPs near the cell membrane; (b) internalization of adsorbed MNPs through the cell membrane into the cytoplasm.

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10. Fig. 9. Magnetization curve of the polycarbonate capsule (a). Dependence of the magnetization value of the maghemite nanoparticle ensemble on the mass of the dried suspension sample with an initial MNP concentration of 4.24 wt.%. The inset shows the experimental data of measuring samples with magnetic moment values ​​less than 0.004 emu, the arrows indicate the lower limits of the possible quantitative assessment of the MNP content (b). All measurements were carried out at 300 K.

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