Dispersion of multi-walled carbon nanotubes in biocompatible dispersants
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- Piret, J., Detriche, S., Vigneron, R. et al. J Nanopart Res (2010) 12: 75. doi:10.1007/s11051-009-9697-8
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Owing to their phenomenal electrical and mechanical properties, carbon nanotubes (CNT) have been an area of intense research since their discovery in 1991. Different applications for these nanoparticles have been proposed, among others, in electronics and optics but also in the medical field. In parallel, emerging studies have suggested potential toxic effects of CNT while others did not, generating some conflicting outcomes. These discrepancies could be, in part, due to different suspension approaches used and to the agglomeration state of CNT in solution. In this study, we described a standardized protocol to obtain stable CNT suspensions, using two biocompatible dispersants (Pluronic F108 and hydroxypropylcellulose) and to estimate the concentration of CNT in solution. CNT appear to be greatly individualized in these two dispersants with no detection of remaining bundles or agglomerates after sonication and centrifugation. Moreover, CNT remained perfectly dispersed when added to culture medium used for in vitro cell experiments. We also showed that Pluronic F108 is a better dispersant than hydroxypropylcellulose. In conclusion, we have developed a standardized protocol using biocompatible surfactants to obtain reproducible and stable multi-walled carbon nanotubes suspensions which can be used for in vitro or in vivo toxicological studies.
KeywordsMulti-walled CNTPluronic F108HPCSonicationCentrifugationHealth safetyNanomedicineEHS
Due to their unique physical and chemical features, nanostructured materials have a wide range of industrial applications. This explains why nanotechnology has become one of the leading technologies over the past 10 years (Stix 2001). For example, the global market for carbon nanotubes (CNT) is predicted to grow up to between $1 billion and $2 billion by 2014 (Thayer 2007). Although the effects of nanoparticles on health and the environment are becoming more of a concern, studies on toxicology and the environmental impact of nanoparticles are still scarce (Colvin 2003).
One of the main issues to investigate the potential toxic effect of isolated CNT on human health using in vitro or in vivo models, is the poor solubility of CNT in water or biological fluids (Buford et al. 2007).
In order to obtain CNT solutions suitable for the in vivo or in vitro studies, there are currently two main ways available. First, CNT surface modifications using water-soluble chemical groups enhance CNT solubilization (Schipper et al. 2008; Wang et al. 2004; Zhao et al. 2008). However, the presence of chemical groups on CNT surface could modify the physico-chemical properties of CNT and render difficult to evaluate the potential toxic effect of the as-received manufactured CNT.
The second way to obtain well-dispersed CNT solutions consist in selection of dispersants for direct dispersion of CNT in water solutions (Jia et al. 2005). The use of dispersants allows dispersion of CNT while avoiding physico-chemical properties modification of the native CNT. However, the direct dispersion is especially difficult in order to achieve a well-dispersed, reproducible, stable CNT suspending solution which is mostly required and important in the biological studies/applications.
For example, there have been several published methodologies for the dispersion of CNT using organic solvents or polymers (Bahr et al. 2001; Holman and Lackner 2006; Thayer 2007). Although these dispersants are very effective at creating stable CNT dispersions, they are often uncompatible with biological experiments due to their intrinsic toxicity. Biocompatible surfactants, like Pluronic F108 (an amphiphilic copolymer composed by hydrophilic ethylene oxide and hydrophobic propylene oxide and used as drug delivery system) and Arabic gum have already been described to be helpful for CNT dispersion (Sayes et al. 2005; Wang et al. 2008). In addition, some suspension agents like hydroxypropylmethylcellulose (HPMC) or hydroxypropylcellulose (HPC), have also been used as dispersant for nanoparticles in different studies (Chen et al. 2006; Meng et al. 2007). However, in these different studies, the protocols for CNT dispersion preparation are not well described and most often only sketchy information about CNT dispersion state is provided (still presence of CNT bundles, only individual CNT in solution, etc.). In this study, we describe a standardized protocol to obtain stable multi-walled carbon nanotubes (MWCNT) suspensions using biocompatible dispersants and to estimate the concentration of MWCNT in solution.
Materials and methods
The size and shape of MWCNT were characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). TEM images were taken using a Tecnai 10 apparatus (Philips) at an acceleration voltage of 80 kV. SEM images were taken using a JEOL 7500F TEM operating at an acceleration voltage of 15 kV. The JEOL 7500F microscope, allowing observation of nanoparticles with a resolution of 1.6 nm, is also equipped with an Energy Dispersive X-ray (EDX) detector for the analysis of the elementary composition (qualitative and quantitative) of nanoparticles. Dry powder of MWCNT was dispersed in ethanol and droplets were placed on TEM grids covered with non-porous or porous formvar before being dried. The surface area of MWCNT was studied by BET analysis.
The size and agglomeration state of MWCNT dispersed in biocompatible surfactants or cell medium were investigated by TEM using the Tecnai 10 apparatus (Philips).
Dispersion of MWCNT
Biocompatible dispersants Pluronic F108 and HPC were purchased from BASF (Ludwigshafen, Germany) and Fargon (Waregem, Belgium), respectively. A total of 1% w/v Pluronic F108 or HPC aqueous solutions were used to obtain homogeneous suspensions of MWCNT. MWCNT solutions were sonicated using a probe sonicator (VC 50T model, power 50 watts and frequency 20 KHz, Sonics & Materials Inc.) at amplitude of 20% for 90 min after 30 min agitation with a magnetic stirrer. MWCNT suspensions were centrifuged at 13000 rpm for 30 min unless stated otherwise.
Culture medium is Dulbecco’s minimal essential medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 5 mL of penicillin–streptomycin/500 mL medium (Biowhittaker Europe).
Estimation of MWCNT concentration
In order to estimate the concentration of MWCNT in suspension after centrifugation, a standard curve was established using non-centrifugated MWCNT-1% Pluronic F108 suspension obtained by sonication. The standard curve was obtained by plotting the absorbance for each standard value against the known concentration. Absorbance of the different standard points was obtained by wavelength scan between 700 and 600 nm using a UV–visible Uvikon spectrophotometer (BRS, Brussels, Belgium). Absorption values of 1% Pluronic F108 or 1% HPC were subtracted from those of MWCNT suspensions. The absorbance values measured between 700 and 600 nm were then used to obtain the average absorbance value for each standard value.
CNT principal characteristics
Average diameter (nm)
Average length (μm)
Surface area (m2/g)
Carbon purity (%)
86.2 ± 0.3
Metal oxide impurities (%)
8.8 ± 2.9
4.4 ± 0.4
0.4 ± 1.5
0.2 ± 1.9
Elementary analysis (using EDX detector) detected catalytic metals (aluminum, iron, cobalt) and oxygen. A Proton Induced X-ray Emission (PIXE) analysis confirmed the presence of aluminum, iron, and cobalt (data not showed).
Other metals may potentially be found depending on the production method and the sensitivity of the analytical method used (see for example Ge et al. 2008), but in any case, if present, they are below 100 ppm level.
MWCNT dispersion in biocompatible surfactants
These data suggest that the protocol followed in this study allows a stable dispersion of MWCNT in biocompatible surfactants.
Estimation of the MWCNT concentration
Dispersion of MWCNT in culture medium
MWCNT are becoming increasingly studied given their possible applications not only in electronics, optics, and mechanical materials but also for biological applications, such as imaging and drug delivery (Lacerda et al. 2006; Martin and Kohli 2003). Thus, it is imperative to assess the possible toxicity of these carbon-based nanostructures. Data already available in literature are controversial. Several toxicological studies have already demonstrated that CNT could exhibit in vitro and in vivo toxicity (Karlsson et al. 2008; Monteiro-Riviere et al. 2005; Muller et al. 2005; Shvedova et al. 2003, 2005), while other authors showed very low or no effect of CNT on cell viability (Davoren et al. 2007; Fiorito et al. 2006; Pulskamp et al. 2007). Different hypotheses could explain these discrepancies. First, MWCNT showed, for example, cytotoxic effects in human epidermal keratinocytes (Monteiro-Riviere et al. 2005) but not in NR8383 macrophage cell line and A549 alveolar cell line (Pulskamp et al. 2007), underlining the fact that cytotoxicity could depend on the cell type used. Another possible explanation may be differences in metal impurities content, which may be as high as 50% in a sample of CNT, depending on the synthesis process (Lam et al. 2006). Moreover, Ge et al. (2008), using neutron activation analysis and inductively coupled plasma mass spectrometry analysis, have recently shown that about 15 different impurity elements could be present in CNT that could influence the potential toxic effect of CNT. However, some studies have shown that effects of MWCNT on cell viability and DNA damage were not dependent on the soluble metals impurities released from MWCNT (Karlsson et al. 2008). Besides cell type and metal impurities, dispersion rate and agglomeration state of CNT in biological fluids and cell culture media could also affect toxicity. In fact, several toxicological studies have been done using agglomerated CNT (Davoren et al. 2007; Karlsson et al. 2008; Pulskamp et al. 2007; Rotoli et al. 2008). The effect of MWCNT on cell viability could be due, in part, to differences in dispersion state (Smart et al. 2006).
In this study, we described the setting of a standardized protocol using biocompatible dispersants to obtain reproducible and stable MWCNT suspensions which can be used for in vitro or in vivo toxicological studies. MWCNT appear to be greatly individualized in these two dispersants without detectable presence of any remaining bundles or agglomerates after sonication and centrifugation. These MWCNT suspensions will be useful to study the biological effect of individualized MWCNT and to compare their effect with previous toxicological data using non-perfectly dispersed MWCNT. Indeed, MWCNT remain well dispersed when added in cell culture medium. Moreover, the use of two different suspension agents will allow comparing the potential effect of dispersants on MWCNT toxicity.
The establishment of a reproducible standard curve using non-centrifugated MWCNT-Pluronic F108 1% solution allows a good estimation of the MWCNT concentration after centrifugation in both dispersants. Lastly, we also showed that the two dispersants do not have the same capability to fully disperse MWCNT, Pluronic F108 providing the best suspensions of isolated MWCNT into water and cell culture medium.
Another interesting point for future investigations is related to the interaction of CNT with the components of cell culture medium. This medium contains various molecules such as amino acids and sugars, as well as fetal calf serum rich in proteins and growth factors necessary for maintaining cells in culture. Although CNT dispersion state in solution could be a important parameter participating to the potential cytotoxicity of CNT, the association of CNT with molecules present in the cell environment could also be of interest when considering the toxicity of CNT in biological fluids.
In conclusion, the reproducible dispersion protocol using different biocompatible surfactants described in this study could be useful to set up a common MWCNT dispersion protocol in order to decrease the discrepancy in toxicological results depending on the variety of strategies regarding CNT dispersion into biological media.
This work is supported by the “Direction Générale des Technologies de la Recherche et de l’Energie” (DGTRE) of the Walloon Region of Belgium (Nanotoxico Project, RW/FUNDP research convention No 516252). O. Toussaint is a Research Associate of the Belgian FNRS.