Carbon nanotubes (CNTs) possess many unique characteristics that have attracted the interest of researchers in a broad range of fields. CNTs have useful electrical, thermal, and mechanical properties, and their material performance can be improved by combination with other materials [1
]. In the medical field, extensive research is underway to develop new CNT biomaterials for use in the treatment and diagnosis of disease [5
]. However, there are concerns about the effect of CNTs on human health [8
]. Since nanomaterials show different physicochemical properties from conventional materials, their effects on human health remain poorly understood. In particular, multi-walled CNTs (MWCNTs) are similar to asbestos in shape and size, raising concern that MWCNTs may be carcinogenic in humans. Safety evaluations of MWCNTs typically use MWNT-7 (Mitsui, Tokyo, Japan), and many reports of in vivo and in vitro experiments have been published. MWNT-7 and other types of MWCNTs show carcinogenicity and cause cytotoxic and inflammatory responses [11
]. However, MWCNTs vary in size and type, and produce different biological responses.
We are developing implants using MWCNTs, tangled-MWCNTs in particular. The safety of tangled-MWCNTs has also been investigated, and they show cytotoxicity but no carcinogenicity [13
]. However, the tangled-MWCNTs that have been investigated were not well dispersed, making it difficult to determine whether the results reflect the effects of nanosized, fibrous MWCNTs or the effects of microsized, agglomerated MWCNTs. We also reported that tangled-MWCNTs, named VGCF-X, showed significant cytotoxicity in the BEAS-2B human bronchial epithelial cell line but little cytotoxicity in the MESO-1 human malignant pleural mesothelioma cell line [18
]. However, at that time we were not able to effectively disperse tangled VGCF-X.
The dispersant is an important factor in dispersion of hydrophobic CNTs. In addition, different dispersants affect not only the size of CNT agglomerates but also biological responses, as we have previously reported [19
]. On the other hand, it is well known that nanoparticles are rapidly coated with various proteins in biological fluids, forming a protein corona [20
]. Therefore, some investigations have used bovine serum albumin (BSA), which is the most abundant protein in serum or plasma, as the dispersant for CNTs [23
]. However, the abundance profile of proteins absorbed to CNTs did not match the protein profile of the serum [26
]. Moreover, differences in biological responses when proteins and surfactants, such as polysorbate 80 (PS) and dipalmitoylphosphatidylcholine (DPPC), are used remain unclear.
In this study, we compared the efficacy of a new sonicator, with improved dispersion efficiency, with conventional sonicators for dispersion for whole tangled MWCNTs, using two dispersants, fetal bovine serum (FBS) and surfactant. Sonicated MWCNTs were evaluated for cytotoxicity on the BEAS-2B cell line, which was previously shown to be more sensitive than the MESO-1 cell line. The findings indicate that tangled MWCNTs can be effectively dispersed by the new sonicator and that the difference in dispersion methods affected cytotoxicity. Moreover, MWCNTs dispersed in FBS produced a different biological response from those dispersed in surfactant.
Sonication and addition of a dispersant are essential to disperse unfunctionalized CNTs, and to evaluate cytotoxicity, because pristine CNTs are hydrophobic and float on water even after vigorous mixing. Moreover, it is well-known that dispersion of tangled MWCNTs is very difficult, even using a proven type of sonicator with high output power. Sonicated CNTs are often not uniformly distributed because supersonic waves show strong directivity and because maximal and minimal points of sound pressure are produced by standing waves. In this study, we tested a new type of sonicator which ameliorates these faults [27
]. This sonication technology can maintain the CNT aspect ratio even after mixing, whereas CNTs were broken during conventional Banbury mixing. In addition, we did not observe a reduction in the size of FT9110 agglomerates with time. This effect could clearly be observed in the size of the FT9110 agglomerates, although the FT9110 sonicated by the three different sonicators did not differ in appearance. Figure S1
shows a light microscopy view of FT9110 in culture medium. Moreover, the quantity of FT9110 that we could confirm visually differed substantially between the PR-1 and the US-1R or W-220 groups, because it was difficult to observe the single fibers produced by the PR-1.
We examined two dispersants in this study: FBS and PS. FBS was selected for its similarity to body fluids. In a previous study, researchers sonicated CNTs in culture medium containing FBS directly, and performed the cytotoxicity test by exposing cells to culture medium containing CNTs. However, sonication causes degradation of macromolecular substances, including proteins. Therefore, we sonicated FT9110 in FBS but without culture medium, then added this solution to the culture medium just before use. The other dispersant, PS, was selected because it is typically used as the dispersant for CNTs [28
]. Our lab has also used PS for in vivo experiments, mainly to avoid provoking an immunoresponse [30
]. Our results showed that PS showed better dispersibility than FBS, using the PR-1. However, because the concentration of the dispersant used was based on our previous experiments using other MWCNTs, and was not optimized for FT9110, the values for FT9110 size are not relevant to further experiments.
Both sonication method and dispersant affected the cytotoxicity of tangled FT9110 individually, and in combination. If we evaluate the cytotoxic effect of the three sonicators using only FBS as the dispersant, the size of the FT9110 agglomerates would lead us to conclude that sonicator type does not affect cytotoxicity. Kim et al. reported that dispersants affect the cytotoxicity of CNTs [33
]. We also found that the biological effects of MWCNTs dispersed in the three dispersants differed substantially [19
]. Although MWCNTs dispersed with gelatin and DPPC induce significant cytotoxicity, carboxymethyl cellulose (CMC) does not show cytotoxicity in BEAS-2B cells. Moreover, we compared the cytotoxicity of gelatin with FBS as a dispersant for MWNT-7 on normal human bronchial epithelial cells, and FBS showed lower cytotoxicity than gelatin [34
]. Liu et al. also reported that single-walled CNT coated with albumin, the most abundant protein in serum, decreased cytotoxicity by reducing cellular uptake [25
]. Here, the reason why cytotoxicity was not affected by the size of FT9110 agglomerates dispersed in FBS may be that uptake of CNTs coated with FBS was reduced. In fact, fewer cells adhered to FT9110 in FBS than in PS.
The cytotoxicity of FT9110 dispersed in PS differed significantly between the PR-1 and US-1R or W-220 groups. FT9110 dispersed by the US-1R and W-220 were endocytosed or adhered. Differences in endocytosis and adhesion seem to depend on agglomerate size. It is worthy of note that some of the cells adhered to FT9110 agglomerates may have been alive, because cytotoxicity increased following exclusion of the FT9110 that were adhered to cells, using the AlamarBlue assay. Excluding CNTs in order to avoid absorption of assay by CNTs, which was reported by Casey et al. [35
], may cause errors in the evaluation of cytotoxicity under these conditions because the FT9110 concentration was lower and the reaction time was shorter than reported by Casey et al. FT9110 dispersed in PS by the PR-1 did not show cytotoxicity, although slight accumulation of FT9110 was observed in the cells. We have used BEAS-2B cells for evaluation of the cytotoxicity of MWCNTs in many previous experiments [36
], and in many cases, cytotoxicity occurred via phagocytosis, following excessive intracellular uptake of MWCNTs. In this study, well-dispersed FT9110 did not stimulate phagocytosis. Individual FT9110 are thinner and more flexible than MWNT-7, the standard material for evaluation of the safety of MWCNTs. Interestingly, re-agglomeration was observed after a time, even if the MWNT-7 were dispersed by a PR-1 sonicator (unpublished data), whereas the dispersed state persisted for a long period after the FT9110 were dispersed once. This indicates that tangled MWCNTs differ from agglomerated MWCNTs. Evaluation of the cytotoxicity of tangled MWCNTs, such as FT9110, should take the intended application into account, and the cytotoxicity of tangled MWCNTs should be evaluated separate from that of single fibers.
4. Materials and Methods
4.1. Suspension and Dispersion of MWCNTs
MWCNT materials were provided by Cnano Technology (FT9110; Santa Clara, CA, USA). The FT9110 were manufactured using a catalytic vapor deposition method; their properties, as provided by the manufacturer, are shown in Table S2
and Figure S2
. The FT9110 were sterilized in an autoclave at 121 °C for 15 min and dried, then 10 mg/mL were vortexed in two dispersants (2% FBS (Biowest, Nuaillé, France) in Dulbecco’s phosphate-buffered saline (DPBS) and 0.1% PS (NOF, Tokyo, Japan) in DPBS) [32
]. Sonication was performed using three different sonicators and their information is shown in Table S3
and Figure S3
The dispersion state of the FT9110 was observed using a transmission electron microscope (TEM; JEOL, Tokyo, Japan). Sonicated FT9110 were diluted to 1 mg/mL with each dispersant and dipped in a microgrid directly. TEM images were captured at 80 kV.
To determine the hydrodynamic size of the agglomerated FT9110, sonicated FT9110 were measured using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The FT9110 were diluted to 1 mg/mL and each measurement was conducted in triplicate.
Dispersed FT9110 at 10 mg/mL were added to cell culture medium at 1/100 volume in each of the following experiments.
4.2. Cell Culture
The BEAS-2B human bronchial epithelial cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). BEAS-2B cells were cultured in Ham’s nutrient mixture F-12 (Nacalai Tesque, Kyoto, Japan) with 10% FBS at 37 °C in a 5% CO2 humidified incubator and passaged twice per week. For each experiment, the cells were seeded at a density of 3 × 105 cells/mL and allowed to adhere for 24 h.
4.3. Cell Viability
Cell viability was assessed using an AlamarBlue assay (alamarBlue® cell viability reagent; Remel, Lenexa, KS, USA). Cells were plated in 96-well plates and incubated for 48 h at 37 °C in culture medium containing 100 μg/mL of FT9110 in a dispersant or in control medium containing only dispersant. After aspiration of the culture medium to exclude the influence of FT9110, 10% AlamarBlue reagent in culture medium was added to each well, where viable cells metabolized the dye for 60 min, resulting in increased fluorescence detected by excitation/emission at 535/590 nm using a plate reader (AF2200; Eppendorf, Hamburg, Germany). Alternatively, AlamarBlue reagent at 10% of the medium volume was simply added to the well without excluding FT9110. Cell viability was calculated as follows: percent cytotoxicity = 100 × experimental value/control value. The media were assayed six times for each treatment condition.
4.4. Observation of Cells by Fluorescence Microscopy
Cells cultured on Cellview glass bottom advanced TC 4 compartments (Griner Bio-one, Frickenhausen, Germany) were exposed to FT9110 for 48 h under the same conditions as described for the cell viability assay. For assessment, cells were stained with bisbenzimide H33342 fluorochrome trihydrochloride (H33342, 10 µg/mL; Nacalai Tesque, Kyoto, Japan) for 1 h before observation. The cells were visualized using an AxioObserverZ1 fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 40× objective lens.
4.5. Statistical Analysis
Data are presented as mean ± S.E. Statistical significance was determined by analysis of variance followed by the Tukey-Kramer method for comparisons between different types of sonicators. P-value < 0.05 was considered statistically significant.