Because MWCNTs are hydrophobic,26
it is important to use dispersants when evaluating MWCNT safety. Therefore, we evaluated the influences of three different dispersants on the viability of cells exposed to a commercial MWCNT material. For dispersants, we selected gelatin and CMC, which are commonly used in pharmaceuticals and food products, as well as DPPC, which is used in experimental lung-exposure conditions.21
Previous safety evaluations of CNTs have used direct suspensions in culture medium or commonly used dispersants (eg, Tween 80, dipalmitoyl lecithin, Arabic gum).29
However, the results of these evaluations were controversial and did not yield clear toxicological information regarding CNTs. It was initially thought that the differences in results were caused by the type of CNT (eg, single-walled versus multi-walled), the included impurities, or differences in cell lines, and little attention was paid to the type of dispersant. Recent reports have described the effects of dispersants on inhibiting the aggregation of CNTs;33
but the differences among the common dispersants are still unclear. No previous reports have compared the effects of different dispersants under the same conditions.
In the present study, we obtained contradictory results of increased and decreased cell viability with the same lot of CNTs and the same cell type; the only difference in experimental conditions was the type of dispersant. These results indicate the possibility that MWCNT safety evaluations are influenced by the type of dispersant. In general, dispersants are amphiphilic and exhibit hydrophilicity and hydrophobicity based on their inherent structure; they disperse hydrophobic substances. In all dispersants, VGCF was dispersed at similar levels after 1 hour, as seen by microscopic observation. At 24 hours, VGCF dispersed and CMC showed no change in agglomeration outside of the cells. By contrast, some of the VGCF dispersed with gelatin or DPPC agglomerated in the culture medium, and most of the single and agglomerated VGCF was internalized into the cells after 24 hours. Moreover, single VGCF fibers showed Brownian motion after 1 hour when dispersed with gelatin or CMC. These observations indicate that differences in dispersants might influence the in vitro response of cells to VGCF. Additionally, the VGCF suspended with PBS could not obtain sufficient suspension under these experimental conditions because of its light weight (results not shown). However, a small amount of agglomerated VGCF in PBS sank to the bottom of the culture dish and was endocytosed by the cells, leading us to surmise that VGCF has the surface characteristics needed for cellular uptake.
Noticeably, there was no distinctive change in the physical characteristics of VGCF dispersed in the three different dispersants, other than agglomerate diameter (). It has been reported that the zeta potential influences the cellular uptake and cytotoxicity of nanomaterials.36
However, those studies measured the zeta potential under different conditions from studies exposing the nanomaterials to cells. In our study, there was no difference in the zeta potential of VGCF dispersed with the three dispersants in culture medium. Moreover, there have been many reports that CNTs enter the cell, and the bioactivity of CNTs is well known.38
These facts indicate that other unknown factors, such as the relationship between CNT receptors on the cell membrane and dispersants, also contribute to the rate of endocytosis. Therefore, dispersants, such as CMC, that inhibit cellular uptake are not suitable for safety assessments of VGCF. However, VGCF with CMC could avoid acute and severe toxicity because of its excellent dispersion. If the mechanism by which CMC suppresses VGCF uptake is clarified and found to apply to the surface-chemical modification of CNT, highly biocompatible CNTs may be developed.26
Recently, many reports have described possible medical applications for surface-modified CNTs.41
We also examined the effect of the dispersion medium on the immune response, which is an index of the critical biological responses to VGCF. SWCNTs have been reported to decrease the proliferation of human lung epithelium in vitro.19
However, the cells used in that report do not internalize SWCNTs. Another study reported that a cytokine assay could not provide accurate measurements because SWCNTs adsorbed cytokines.18
However, BEAS-2B cells that internalized VGCF in gelatin or in DPPC exhibited increased IL-6 and IL-8 secretion, whereas cytokine secretion was not altered when the VGCF in CMC were not internalized. Cytokine secretion, which is an indicator of an inflammatory response, is increased in the bronchoalveolar lavage fluid of mice exposed to CNTs.43
Our results indicate that VGCF uptake is critical for cytokine secretion, which is consistent with other in vivo studies.
In the present study, the amount of VGCF uptake was represented as the relative ratio compared with the VGCF-free experimental condition. As a result, the uptake amounts of VGCF in gelatin were higher than those of VGCF in DPPC at 1 hour after VGCF exposure. At that time, the amounts of VGCF on the bottom of the culture dish were similar for all three dispersants.
The dispersant might also affect the uptake rate of VGCF into the cells. The intracellular amount of VGCF in gelatin was increased, compared with other dispersants; consequently, the intracellular VGCF inhibited cell proliferation and decreased cell viability to less than 50%. The doubling times of BEAS-2B and MESO-1 cells are approximately 28 hours and 26 hours, respectively, and the cell count had almost doubled at the time of the alamarBlue® assay in this experiment. Thus, the cell viability of less than 50% represented the cytotoxic effect of VGCF. In the case of DPPC, we think that the cells were proliferating during VGCF uptake, and consequently, the concentration of VGCF per cell was reduced. Therefore, the cell viability was maintained at approximately 80% by VGCF in DPPC as a result of the relatively small VGCF uptake. In contrast, in the experiment with VGCF in gelatin and BEAS-2B cells, the uptake ratio at 80% cell viability was approximately 1.8, which was similar to VGCF in DPPC. This result suggests that the cell viability of VGCF was related to the intracellular concentration of VGCF, not to the type of dispersant.
Moreover, the relationship between cell viability and cell differences might not be significant because the relative uptake ratio was approximately 2.1 at the IC50 of VGCF in both types of cells. However, in the microscopic images, it seemed that the absolute amount of intracellular VGCF differed between BEAS-2B cells and MESO-1 cells. The relative ratio of VGCF seemed to represent an absolute quantity of VGCF versus the intracellular capacity, because the SSC of flow cytometry represents the inner complexity of the cells based on the shape of the nucleus, the amount and type of cytoplasmic granules, or the membrane roughness. In fact, the cell volume of MESO-1 cells was about 2.3 times larger than that of BEAS-2B cells. This value was almost the same as that of the IC50 concentration.
We performed the AB assay and measured SSC simultaneously, under the same experimental conditions. When the starting cell density was changed, the SSC value and cell viability at one VGCF concentration was also altered, but the SSC value at IC50 was constant.
In conclusion, we found that the biological response to VGCF in vitro is influenced by the dispersant. The variety of dispersant affects the uptake rate and cell proliferation rate. In contrast, VGCF induced cytotoxicity and inflammatory reactions based on the intracellular concentration of VGCF, not based on cell differences. More detailed studies are needed to elucidate the exact biological mechanisms involved.