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Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids.

Yakymchuk OM, Perepelytsina OM, Dobrydnev AV, Sydorenko MV - Nanoscale Res Lett (2015)

Bottom Line: Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect.The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples.Our results could be useful for the control of cell growth in three-dimensional culture. 61. 46 + w; 61.48 + c; 61.48De; 87.15-v; 87.64-t.

View Article: PubMed Central - PubMed

Affiliation: Department for Biotechnical Problems of Diagnostic, Institute for Problems of Cryobiology and Cryomedicine of NAS Ukraine, 42/1 Nauky str., 03028 Kiev, Ukraine.

ABSTRACT

Abstract: This paper describes the impact of different concentrations of single-walled carbon nanotubes (SWCNTs) on cell viability of breast adenocarcinoma, MCF-7 line, and formation of multicellular tumor spheroids (MTS). Chemical composition and purity of nanotubes is controlled by Fourier transform infrared spectroscopy. The strength and direction of the influence of SWCNTs on the tumor cell population was assessed by cell counting and measurement of the volume of multicellular tumor spheroids. Effect of SWCNTs on the formation of multicellular spheroids was compared with the results obtained by culturing tumor cells with ultra dispersed diamonds (UDDs). Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect. The increasing of SWCNTs concentration to 100 to 200 μg/ml stimulated proliferation of tumor cells, especially in suspension fractions. The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples. In result, the median volume of MTS after cultivation with SWCNTs at 100 to 200 μg/ml concentrations is 3 to 5 times greater than that in samples which were incubated with the UDDs and is 2.5 times greater than that in control cultures. So, if SWCNTs reduced cell adhesion to substrate and stimulated formation of tumor cell aggregates volume near 7 · 10(-3) mm(3), at the same time, UDDs reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids volume no more than 4 · 10(-3) mm(3). Our results could be useful for the control of cell growth in three-dimensional culture.

Pacs: 61. 46 + w; 61.48 + c; 61.48De; 87.15-v; 87.64-t.

No MeSH data available.


Related in: MedlinePlus

Multicellular tumor spheroids culture at different concentrations of SWCNTs. (A) Control. (B) 12.5 μg/ml. (C) 25 μg/ml. (D) 50 μg/ml. (E) 100 μg/ml. (F) 150 μg/ml. (G) 200 μg/ml. ×80 magnification.
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Fig4: Multicellular tumor spheroids culture at different concentrations of SWCNTs. (A) Control. (B) 12.5 μg/ml. (C) 25 μg/ml. (D) 50 μg/ml. (E) 100 μg/ml. (F) 150 μg/ml. (G) 200 μg/ml. ×80 magnification.

Mentions: We discovered several physical, chemical, and biological trends. First, at low concentrations of SWCNTs (12.5 and 25.0 μg/ml), the highest number of small (10−4 mm3) tumor cell aggregates was formed (Figure 4). In the process of increasing concentrations of SWCNTs, we observed the growth of the volume of MTS and decrease of the number of it. So, small concentrations of SWCNTs stimulated cells to migrate into suspension and generate MTS with volume from 0.1 to 0.3°10−4 mm3. Increasing concentrations of SWCNTs from 50 to 200 μg/ml led to decrease the number of cell spheroids but enlarged their volume from 7 to 25°10−2 mm3. This trend was particularly notable at concentration of SWCNTs at 200 μg/ml. The viability of tumor cells in suspension fraction at 200 μg/ml of SWCNTs was even higher than that in the control. As a result, the volume of MTS was the largest at 200 μg/ml of SWCNTs. A positive correlation between concentration of SWCNTs and volume of MTS was confirmed by the statistical analysis. The Pearson’s coefficient in this series of experiments was 0.89. It means a very close positive correlation between concentration of SWCNTs and volume of MTS. So, we suggested that SWCNTs promoted cell migration, viability in suspension, and aggregation in spheroids. According to the literature, SWCNTs may act as an artificial extracellular matrix [20]. For example, Firkovska with colleges demonstrated that mouse fibroblast cells were able to grow on nanostructured carbon nanotubes substrates. In a recent series of experiments, they showed that the quality of the nanotubes can significantly stimulate the growth and proliferation of mammalian cells. These results showed that highly ordered arrays of carbon nanotubes can be used to manage and control the growth of mammalian cells [21]. Our data can be viewed from two perspectives. Firstly, incubation of tumor cells with certain concentrations of nanotubes clearly leads to tumor progression. This happens after stimulation of cell detachment from the substrate and the formation of large numbers of small tumor spheroids (micrometastases). As reported in the literature, cell aggregate of 0.1 mm in diameter already ables to migrate into the blood stream and lymphoid system and to form a secondary tumor [22]. In connection with this, oncogenic activity of the SWCNTs under certain conditions and prerequisites can cause cancer and should be the object of attention of scientists [23-25]. So nanostructured materials are perspective for creation of biological sensors, carriers of drugs, and diagnostic elements but should be used very carefully [26-28]. The effect of nanotubes on the development of tumor process can occur at the level of cell-to-cell interaction, with extracellular matrix and stoma structures. On the other hand, according to our research and literature data, chemical and physical properties of SWCNTs demonstrated low cytotoxic effect on the intracellular structures [11,29,30]. Therefore, the usage of SWCNTs as basis for intensive growth of cells in suspension, three-dimensional culture, and tissue-like structures seems very promising. Proof of this is in well-known biotechnology experiments which describe fibroblast-like and endothelial cells growth on thin films of SWCNTs [19]. Second, the trend influences the chemical properties of nanomaterials on the formation of MTS. To compare the effects of SWCNTs and UDDs on the formation of cell aggregates, we calculated the median of volume of MTS at appropriate concentrations of these nanostructures (Figure 5). The formation of MTS is very important because tumor spheroids are well-known model of avascular stage of tumor growth and formation of micrometastases. Another feature that caught our attention relates to the physical characteristics of NM, which affect the relationship between cells and carbon structure. For comparison, we used carbon structures called UDDs. We described their properties and effects on cells in another paper [11]. So, we compared the sizes of MTS which were formed in the presence of SWCNTs and UDDs. We found that UDDs stimulated the formation of MTS at concentrations of 12.5 to 50 μg/ml. Therefore, SWCNTs created more favorable conditions for cell spheroid growth at concentrations 100 to 200 μg/ml. At the same time, the median of MTS volume was larger in the presence of SWCNTs than that in the presence of UDDs at all concentrations (Figure 6). We calculated Pearson’s coefficient for UDDs. It was −0.81. So, UDDs demonstrated inverse correlation between the size of cell aggregates and concentration of UDDs. The explanation of this phenomenon is in the size of nanostructured aggregates, their effect on cell adhesion to the substrate, and physical properties of cells and NM. Nowadays, there are a lot of papers devoted to biological usage of SWCNTs and its promising prospects. But mechanisms of interaction of SWCNTs with living cells and tissues still remain unclear. It should be noted that use of SWCNTs in biological systems encounters on a number of difficulties. For example, the hydrophobic nature of SWCNTs as well as all carbon nanomaterials promotes fast aggregation of SWCNTs in physiological solutions. At the same time, the functionalization of SWCNTs provides better solubility and changes the electrochemical properties of substances. For example, according to the literature, zeta potential of SWCNTs is about −10 to −20 mV in pH range 6.0-8.0 [29]. It should be remembered that zeta potential of a healthy cell is about −20.5 mV. Processing SWCNTs in surfactant, for example, cetyltrimethylammonium bromide (CTAB) leads to change in zeta potential from −19.38 to 49.51 mV and better solubility [30]. According literature data three-dimensional and chemical structure of SWCNTs is quite complicated [31,32]. Our assay of SWCNTs FTIR spectra testified the presence of a small amount of O-H and N-H groups (lines in 3,432 nm area). Low power of functionalization and high removal of impurities provided aggregation of SWCNTs and formation of cohesive centers for tumor cells in three-dimensional culture. And the same negative zeta potential of cells and SWCNTs prevented the establishment of strong focal cellular contacts with cells and carbon aggregates. We observed a different trend for the UDDs. In our previous studies, we have demonstrated that the UDDs stimulated the formation of multicellular tumor spheroids at low concentrations. FTIR assay of UDDs demonstrated a presence of a large number of functional groups on its surface, and zeta potential was about +40 mV. This leads to a weakening of cell-substrate contact, as we observed at low concentrations of the UDD [11]. But this effect is not sufficient for the formation and survival of large cell aggregates in the suspension. We assumed that the differences in cell-to-cell interactions in culture and those with considered nanostructures can be explained by differences in electrochemical properties of nanomaterial substances and cells.Figure 4


Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids.

Yakymchuk OM, Perepelytsina OM, Dobrydnev AV, Sydorenko MV - Nanoscale Res Lett (2015)

Multicellular tumor spheroids culture at different concentrations of SWCNTs. (A) Control. (B) 12.5 μg/ml. (C) 25 μg/ml. (D) 50 μg/ml. (E) 100 μg/ml. (F) 150 μg/ml. (G) 200 μg/ml. ×80 magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig4: Multicellular tumor spheroids culture at different concentrations of SWCNTs. (A) Control. (B) 12.5 μg/ml. (C) 25 μg/ml. (D) 50 μg/ml. (E) 100 μg/ml. (F) 150 μg/ml. (G) 200 μg/ml. ×80 magnification.
Mentions: We discovered several physical, chemical, and biological trends. First, at low concentrations of SWCNTs (12.5 and 25.0 μg/ml), the highest number of small (10−4 mm3) tumor cell aggregates was formed (Figure 4). In the process of increasing concentrations of SWCNTs, we observed the growth of the volume of MTS and decrease of the number of it. So, small concentrations of SWCNTs stimulated cells to migrate into suspension and generate MTS with volume from 0.1 to 0.3°10−4 mm3. Increasing concentrations of SWCNTs from 50 to 200 μg/ml led to decrease the number of cell spheroids but enlarged their volume from 7 to 25°10−2 mm3. This trend was particularly notable at concentration of SWCNTs at 200 μg/ml. The viability of tumor cells in suspension fraction at 200 μg/ml of SWCNTs was even higher than that in the control. As a result, the volume of MTS was the largest at 200 μg/ml of SWCNTs. A positive correlation between concentration of SWCNTs and volume of MTS was confirmed by the statistical analysis. The Pearson’s coefficient in this series of experiments was 0.89. It means a very close positive correlation between concentration of SWCNTs and volume of MTS. So, we suggested that SWCNTs promoted cell migration, viability in suspension, and aggregation in spheroids. According to the literature, SWCNTs may act as an artificial extracellular matrix [20]. For example, Firkovska with colleges demonstrated that mouse fibroblast cells were able to grow on nanostructured carbon nanotubes substrates. In a recent series of experiments, they showed that the quality of the nanotubes can significantly stimulate the growth and proliferation of mammalian cells. These results showed that highly ordered arrays of carbon nanotubes can be used to manage and control the growth of mammalian cells [21]. Our data can be viewed from two perspectives. Firstly, incubation of tumor cells with certain concentrations of nanotubes clearly leads to tumor progression. This happens after stimulation of cell detachment from the substrate and the formation of large numbers of small tumor spheroids (micrometastases). As reported in the literature, cell aggregate of 0.1 mm in diameter already ables to migrate into the blood stream and lymphoid system and to form a secondary tumor [22]. In connection with this, oncogenic activity of the SWCNTs under certain conditions and prerequisites can cause cancer and should be the object of attention of scientists [23-25]. So nanostructured materials are perspective for creation of biological sensors, carriers of drugs, and diagnostic elements but should be used very carefully [26-28]. The effect of nanotubes on the development of tumor process can occur at the level of cell-to-cell interaction, with extracellular matrix and stoma structures. On the other hand, according to our research and literature data, chemical and physical properties of SWCNTs demonstrated low cytotoxic effect on the intracellular structures [11,29,30]. Therefore, the usage of SWCNTs as basis for intensive growth of cells in suspension, three-dimensional culture, and tissue-like structures seems very promising. Proof of this is in well-known biotechnology experiments which describe fibroblast-like and endothelial cells growth on thin films of SWCNTs [19]. Second, the trend influences the chemical properties of nanomaterials on the formation of MTS. To compare the effects of SWCNTs and UDDs on the formation of cell aggregates, we calculated the median of volume of MTS at appropriate concentrations of these nanostructures (Figure 5). The formation of MTS is very important because tumor spheroids are well-known model of avascular stage of tumor growth and formation of micrometastases. Another feature that caught our attention relates to the physical characteristics of NM, which affect the relationship between cells and carbon structure. For comparison, we used carbon structures called UDDs. We described their properties and effects on cells in another paper [11]. So, we compared the sizes of MTS which were formed in the presence of SWCNTs and UDDs. We found that UDDs stimulated the formation of MTS at concentrations of 12.5 to 50 μg/ml. Therefore, SWCNTs created more favorable conditions for cell spheroid growth at concentrations 100 to 200 μg/ml. At the same time, the median of MTS volume was larger in the presence of SWCNTs than that in the presence of UDDs at all concentrations (Figure 6). We calculated Pearson’s coefficient for UDDs. It was −0.81. So, UDDs demonstrated inverse correlation between the size of cell aggregates and concentration of UDDs. The explanation of this phenomenon is in the size of nanostructured aggregates, their effect on cell adhesion to the substrate, and physical properties of cells and NM. Nowadays, there are a lot of papers devoted to biological usage of SWCNTs and its promising prospects. But mechanisms of interaction of SWCNTs with living cells and tissues still remain unclear. It should be noted that use of SWCNTs in biological systems encounters on a number of difficulties. For example, the hydrophobic nature of SWCNTs as well as all carbon nanomaterials promotes fast aggregation of SWCNTs in physiological solutions. At the same time, the functionalization of SWCNTs provides better solubility and changes the electrochemical properties of substances. For example, according to the literature, zeta potential of SWCNTs is about −10 to −20 mV in pH range 6.0-8.0 [29]. It should be remembered that zeta potential of a healthy cell is about −20.5 mV. Processing SWCNTs in surfactant, for example, cetyltrimethylammonium bromide (CTAB) leads to change in zeta potential from −19.38 to 49.51 mV and better solubility [30]. According literature data three-dimensional and chemical structure of SWCNTs is quite complicated [31,32]. Our assay of SWCNTs FTIR spectra testified the presence of a small amount of O-H and N-H groups (lines in 3,432 nm area). Low power of functionalization and high removal of impurities provided aggregation of SWCNTs and formation of cohesive centers for tumor cells in three-dimensional culture. And the same negative zeta potential of cells and SWCNTs prevented the establishment of strong focal cellular contacts with cells and carbon aggregates. We observed a different trend for the UDDs. In our previous studies, we have demonstrated that the UDDs stimulated the formation of multicellular tumor spheroids at low concentrations. FTIR assay of UDDs demonstrated a presence of a large number of functional groups on its surface, and zeta potential was about +40 mV. This leads to a weakening of cell-substrate contact, as we observed at low concentrations of the UDD [11]. But this effect is not sufficient for the formation and survival of large cell aggregates in the suspension. We assumed that the differences in cell-to-cell interactions in culture and those with considered nanostructures can be explained by differences in electrochemical properties of nanomaterial substances and cells.Figure 4

Bottom Line: Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect.The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples.Our results could be useful for the control of cell growth in three-dimensional culture. 61. 46 + w; 61.48 + c; 61.48De; 87.15-v; 87.64-t.

View Article: PubMed Central - PubMed

Affiliation: Department for Biotechnical Problems of Diagnostic, Institute for Problems of Cryobiology and Cryomedicine of NAS Ukraine, 42/1 Nauky str., 03028 Kiev, Ukraine.

ABSTRACT

Abstract: This paper describes the impact of different concentrations of single-walled carbon nanotubes (SWCNTs) on cell viability of breast adenocarcinoma, MCF-7 line, and formation of multicellular tumor spheroids (MTS). Chemical composition and purity of nanotubes is controlled by Fourier transform infrared spectroscopy. The strength and direction of the influence of SWCNTs on the tumor cell population was assessed by cell counting and measurement of the volume of multicellular tumor spheroids. Effect of SWCNTs on the formation of multicellular spheroids was compared with the results obtained by culturing tumor cells with ultra dispersed diamonds (UDDs). Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect. The increasing of SWCNTs concentration to 100 to 200 μg/ml stimulated proliferation of tumor cells, especially in suspension fractions. The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples. In result, the median volume of MTS after cultivation with SWCNTs at 100 to 200 μg/ml concentrations is 3 to 5 times greater than that in samples which were incubated with the UDDs and is 2.5 times greater than that in control cultures. So, if SWCNTs reduced cell adhesion to substrate and stimulated formation of tumor cell aggregates volume near 7 · 10(-3) mm(3), at the same time, UDDs reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids volume no more than 4 · 10(-3) mm(3). Our results could be useful for the control of cell growth in three-dimensional culture.

Pacs: 61. 46 + w; 61.48 + c; 61.48De; 87.15-v; 87.64-t.

No MeSH data available.


Related in: MedlinePlus