Teste Nanosof

SCIENTIFIC REPORT CONCERNING THE ANTIOXIDAT EFFECTS OF NANOSOF POWDER SUSPENSION ON DIFFERENT CELL LINES EXPOSED TO EXOGENOUS FREE OXYGEN RADICALS

The aim of the study was to assess the protective effect of liquid suspension of Nanosof powder on several cell cultures exposed to exogenous free oxygen radicals via incubation with hydrogen peroxide (H2O2).

Cell lines used in this study

* bEnd 3 is a standardized immortalized cell line derived from endothelial cells isolated from the brain, particularly cerebral cortex, of Mus musculus (common mouse);

* Bv2 is a retrovirus-immortalized microglia cell line primarily obtained from mouse (Mus musculus) brain;

* HEK293T (human embryo kidney) is a human diploid cell line extracted from embryonic kidney (metanephros), expressing the SV40 T retroviral antigen; this cell line stably expresses the main transmembrane subunit of the human cardiac ion channel hERG1, in combination with a geneticin (G-418) resistance element.

Nanosof is an oxygenated fullerene chemically heterogenous product (over 80% elemental oxygen content) obtained by original methods from carbon products of animal origin. The product was previously tested in multiple in vitroin vivo, and clinical trials, and was shown to exert several remarkable therapeutic effects, most notably an increased scavenging effect on free oxygen radicals, and potentiating effects on the antioxidant properties of other antioxidants of natural origin. These properties are exploited in the commercially available product Nanoxyn alpha, a product approved by FDA as food supplement and delivered on the US market (more data available on the company website https://www.nanoxyn.ro/).

Preparation of the Nanosof powder suspension used for these experiments was achieved by mixing 1 mg of pure powder (red rusty color and paramagnetic properties) per 1 ml of cell culture medium in a plastic tube, followed by moderate centrifuging (either 1 min at 3200 rpm into an Eppendorf microcentrifuge or 1000 g for 10 min in a Heraeus Labofuge 400R centrifuge with swing-out rotor). The pellet was discarded, while the supernatant was collected with a sterile syringe and subsequently filtered through 0.22 m filters (Millipore Millex®GP Filter units with Millipore Express® PES membranes). Prior to centrifuging, the Nanosof primary suspension was vortexed. For the bEnd3 cell line, the cell culture medium was Advanced DMEM – Gibco 12491-015 supplemented with 10% fetal bovine serum (Sigma F2442 – US origin, sterile-filtered, endotoxin and hemoglobin-free), 1% Penicillin/Streptomycin (Gibco 15140-122), and 2 mM L-Glutamine (from Lonza #BE17-605E stock solution). For the other two cell lines, the medium was low glucose DMEM (Sigma D5523), resuspended according to manufacturer’s instructions, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine (from Sigma G7513 stock solution)

Cell culture protocol. All three cell lines were detached from 25 cm2 angled cell culture flasks (Nunclon® Surface) following trypsin-EDTA treatment (3 min at 37 C) with sterile Pasteur pipettes, centrifuged briefly (5 min at 1000 g), resuspended in control cell culture medium, and cell density was counted with a Bürker-Türk chamber and adjusted such as to plate 5000 cells per well in 100 µl total volume in a 96-well cell culture plate with flat bottom. Each cell line was disposed in 12 wells following a predesigned pattern (see below). The cells were grown overnight in a humidified incubator (37 °C, 5% CO2).

Note. We could have attempted a higher cell density, e.g. 10,000 cells/well.

Treatment applied to the cultured cell lines

All experimental conditions were applied in triplicate, i.e. in three distinct wells on the 96-well plate, and absorbance read-outs were averaged over these three wells.

Day 1. After seeding the cells in 12 wells for each cell line in a total volume of 100µl/well, the plate was left overnight in the incubator at 37°C. 3 wells were used for background readouts, and were filled with 100µl of medium each, devoid of cells.

Day 2. During the following days, the 12 cell-containing wells for each cell line were emptied and filled with different contents, according to the pattern specified in Figure 1. The cells were kept in the incubator at 37 °C for 24 hours.

The following conditions were tested in triplicate for each cell line:

– 3 wells with cells treated for 24 hours with normal cell culture medium (no treatment added);

– 3 wells with cells treated for 24 hours with 100µM H2O2;

– 3 wells with cells treated for 24 hours with 100µM H2O2 with Nanosof suspension;

– 3 wells with cells treated for 24 hours with Nanosof suspension only;

– 3 wells with cell culture medium without cells (for background spectrophotometer readouts), where the medium was replaced with a fresh normal medium.

H2O2 (-)

H2O2 (100 µM)

1

2

3

4

5

6

7

8

9

10

11

12

Nanosof (-)

A

Nanosof (+)

B

Nanosof (-)

C

Bg

Bg

Bg

D

E

F

G

H

Figure 1. Experimental cell culture pattern in 96-well plate for the MTS cell viability test

Day 3. After 24 hours of incubation in specific conditions, 20 µl of MTS solution were added to each well, and the plate was left at room temperature, in sterile conditions. Absorbance at 490 nm (for optical density – OD) and at 650 nm (for background) was read with a plate spectrophotometer at 1 h, 2 h, and 4 h since MTS addition. After 3 hours, the content of each well was mixed by gentle aspiration and release with a laboratory pipette pipette 5-fold.

MTS represents an in vitro cell viability test based on an organic tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and a coupling reagent via electron transfer [phenazine ethosulfate; PES], featuring high stability, and thus able to combine with MTS to form a stable solution. Spectrophotometric absorbance at 490 nm, measured at 1-4 hours following application in a well, is proportional to the number of viable cells.

Results

Hydrogen peroxide (H2O2) is a well-known and widely used system for exposure of biological systems to exogenous free oxygen radicals. Our results for all three cell lines tested in this experiment indicate that Nanosof suspension effectively neutralizes the toxic effect on cell viability of hydrogen peroxide, without being toxic for the cells per se. Figure 2 illustrates MTS absorbance values (mean of 3 wells for each tested condition) assessed at 4 hours, and mean values are listed in the following table. The absorbance values were processed as follows: read-outs at 490 nm were background-subtracted with average values of background wells at 490 nm; read-outs at 650 nm were background-subtracted with average values of background wells at 650 nm; background-corrected absorbance values at 610 nm for each well were subtracted from background-corrected absorbance values at 490 nm for the same well.

Figure 2. Effect of Nanosof suspension on background cell viability and following exposure to H2O2 for all three tested cell lines (absorbance at 490 nm at 4 h following exposure to MTS, mean of three wells/experimental condition)

bEnd3

N(-)

N(+)

Bv2

N(-)

N(+)

HEK

N(-)

N(+)

H2O2(-)

0.7942

0.739367

H2O2(-)

0.689007

0.709007

H2O2(-)

0.161007

0.140007

H2O2(+)

0.355167

0.6456

H2O2(+)

0.345007

0.597007

H2O2(+)

0.009007

0.082007

Conclusions

1. Nanosof suspension applied in the presence of H2O2 exerted beneficial effects, possibly via scavenging or otherwise neutralizing exogenous free oxygen radicals, in all three tested cell lines.

2. Nanosof suspension was not toxic and did not significantly influence cell viability for all three tested cell lines.

3. The exogenous free radical exposure model chosen for this experiment was adequate, resulting in significant decreases in cell viability for all three tested cell lines.

Perspectives

Nanosof suspension could be useful in cell culture experiments for enhancing cell viability and limiting deleterious effects of free radicals. We could use it for example in the cell culture medium of Cor.4U® human induced pluripotent stem cell-derived cardiomyocytes to limit toxic effects of free oxygen radicals on various cellular components, for example on L-type calcium channels. We will seek to perform further tests concerning effects of Nanosof suspension on cell migration capacity.

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