Effect of MWCNT surface and chemical modification on in vitro cellular response
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- Fraczek-Szczypta, A., Menaszek, E., Syeda, T.B. et al. J Nanopart Res (2012) 14: 1181. doi:10.1007/s11051-012-1181-1
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The aim of this study was to evaluate the impact of multi-walled carbon nanotubes (MWCNTs with diameter in the range of 10–30 nm) before and after chemical surface functionalisation on macrophages response. The study has shown that the detailed analysis of the physicochemical properties of this particular form of carbon nanomaterial is a crucial issue to interpret properly its impact on the cellular response. Effects of carbon nanotubes (CNTs) characteristics, including purity, dispersity, chemistry and dimension upon the nature of the cell environment–material interaction were investigated. Various techniques involving electron microscopy (SEM, TEM), infrared spectroscopy (FTIR), inductively coupled plasma optical emission spectrometry, X-ray photoelectron spectroscopy have been employed to evaluate the physicochemical properties of the materials. The results demonstrate that the way of CNT preparation prior to biological tests has a fundamental impact on their behavior, cell viability and the nature of cell–nanotube interaction. Chemical functionalisation of CNTs in an acidic ambient (MWCNT-Fs) facilitates interaction with cells by two possible mechanisms, namely, endocytosis/phagocytosis and by energy-independent passive process. The results indicate that MWCNT-F in macrophages may decrease the cell proliferation process by interfering with the mitotic apparatus without negative consequences on cell viability. On the contrary, the as-prepared MWCNTs, without any surface treatment produce the least reduction in cell proliferation with reference to control, and the viability of cells exposed to this sample was substantially reduced with respect to control. A possible explanation of such a phenomenon is the presence of MWCNT’s agglomerates surrounded by numerous cells releasing toxic substances.
KeywordsCarbon nanotubes Surface characterisation Functionalisation In vitro investigation
Nanomaterials as the foundation of nanotechnology are increasing in importance due to the needs of different branches of industry and medicine for modern and improved performance products and medical devices. Increasingly frequently, however, one asks oneself whether in addition to the benefits of nanotechnology, it may also be harmful for health. This question relates primarily to nanoparticles such as carbon nanotubes (CNTs), production of which since 1991 has increased rapidly. The interest in CNTs is mainly associated with their unique properties, which make them attractive options in various consumer, medical, and industrial applications (Schrand et al. 2007; Xu et al. 2008; Harrison and Atala 2007; Kim et al. 2011). Opinions on the biocompatibility of CNTs in vitro and in vivo environments are not, however, unequivocal. Some researchers have compared them to the negative effects of asbestos fibres, ordering special care during handling or disqualifying them completely from further use (Jaurand et al. 2009; Poland et al. 2008). Particular caution is advised during contact of CNTs with skin and the respiratory tract (Tong et al. 2009). Other researchers in turn have indicated that nanotubes are biocompatible and have a positive impact on cell growth and proliferation and, therefore, may be used in tissue engineering (Shi et al. 2007; Matsumoto et al. 2007; Fraczek et al. 2008). Additionally, due to their mobility potential in living systems, they may be successfully used as novel drug delivery systems for therapy and diagnosis (Xu et al. 2008; Menard-Moyon et al. 2010). In contrast to observations that nanotubes tend to accumulate in tissues and organs, other experiments have shown that more than 95 % of CNTs are released in the urine from the body within a few hours (Liu et al. 2009; Lacerda et al. 2008; Guoa et al. 2007) or are degraded (Liu et al. 2010). Analysis of the available literature shows both the beneficial and the adverse impact of nanotubes on a living organism and suggests that both parties are right. Moreover, taking into account the diversity of CNTs resulting primarily from methods of their manufacture, the catalysts used, the synthesis conditions and a variety of methods used for evaluation of their toxicity, it is difficult to agree with either view. Many studies indicate that biocompatibility of CNTs in both in vivo and in vitro studies may be attributed to various factors, including their lengths, functionality, their concentration, duration in the living body, catalyst impurity, agglomeration and even the dispersants used to dissolve the nanotubes (Kagan et al. 2005, 2006; Bianco et al. 2005; Sato et al. 2005; Wick et al. 2007; Aillon et al. 2009; Raja et al. 2007; Coccini et al. 2010; Fiorito et al. 2009). It is also important to develop and validate methods to evaluate the toxicity of nanoparticles to compare properly the experimental results between research institutions. Most aspects of CNT toxicity still remain inadequately identified and further long-term research is required.
The objective of this study is to evaluate the impact of MWCNTs differing in surface chemistry, purity and dispersion-degree on cellular response. The effect of differently prepared MWCNTs on the viability of macrophages (RAW 264.7) was analysed.
Materials and methods
Three types of as-prepared and functionalised MWCNTs were used in this study. The as-prepared MWCNTs examined in this study were provided by NanoAmorUSA. The pristine MWCNTs had diameters in the range of 10–30 nm and were 1–2 μm long.
The next step after the chemical oxidation of the MWCNT-F was their functionalisation in ethylenediamine (C2H4(NH2)2). Amino-functionalisation of CNTs was performed to verify their influence on cell response. The literature states that the functionalisation of CNTs significantly reduces their cytotoxicity and improves cell growth (Vukovic et al. 2010; Hu et al. 2004).
In the first step, the MWCNT-F were treated in a mixture of SOCl2 (thionyl chloride): dimethylformamide (DMF) (20:1) for 24 h at 70 °C, followed by cooling them to room temperature and finally the nanotubes were washed several times using tetrahydrofuran (THF) to remove excess SOCl2. The aim of this step was to generate acyl chloride groups and facilitate functionalisation of CNTs with amines. Subsequently, such prepared CNTs were treated with ethylenediamine (C2H4(NH2)2) for 48 h at a 95 °C. After this treatment, the CNTs were washed in ethanol and dried under vacuum. CNTs prepared in such a way were denoted as MWCNT-NHs. The morphology of MWCNTs before and after chemical oxidation was analysed using transmission electron microscopy (TEM) (Tecnai G2 F20 (200 kV) and Joel). The absolute zeta potential (ζ) and size distribution of CNTs before and after acid oxidation were performed in phosphate buffer (PBS), using combination of electrophoresis and the LDV technique (Laser Doppler Velocimetry, Malvern Zetasizer Nano ZS) in the range of the particle size from 5 nm to 10 μm, with the laser light source of wavelength λ = 520 nm.
The degree of purification of CNTs was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) (Multiwave 3000, Perkin Elmer Co.). Evaluation of the functionalisation of CNTs was performed using Fourier transformation infrared spectroscopy (FTIR) (Bio-Rad FTS60 V spectrometer). The transmission of FTIR spectra was registered in the range of 800–3800 cm−1 using KBr pellets.
The study of composition and chemical state of selected elements was made using X-ray photoelectron spectroscopy (XPS) (Vacuum Systems Workshop Ltd., England). Depth of analysis was about 5 nm. Mg Kα X-ray radiation with 200 W energy was used as the excitation source. Electron energy analyzer was set to FAT mode with pass energy 22 eV. The shift of the binding energy due to surface charging effect was calibrated by assuming binding energy of C1s to be always 284.6 eV.
The murine macrophage RAW 264.7 cell line (ATCC, GB) was used in this study. The cells were cultured in 75 cm2 tissue culture flasks (Nunc, Denmark) in Dulbecco’s modified Eagle’s medium (DMEM; PAA, Austria) supplemented with antibiotics (penicillin G 100 U/ml, streptomycin 10 μg/ml (Sigma-Aldrich, Germany)) and 10 % bovine foetal serum (PAA, Austria). The flasks of cultured cells were incubated at 37 °C in humidified 95 % air and 5 % CO2. Cells were routinely processed by harvesting using a cell scraper and replicated in tissue culture flasks at a ratio of 1:5.
Before incubation with cells (in vitro tests), each type of CNTs was sonicated for 2 min using a tip sonicator (PALMER INSTRUMENTS, Model: CP 130 PB, 130 W power, 20 kHz) in PBS, with a dual concentration of CNT 38 and 80 μg/ml, respectively. Subsequently, CNTs were sterilised by the UV method for 0.5 h. The interaction of nanotubes with RAW 264.7 macrophages was observed using an inverted microscope (Olympus CKX41, Germany) and scanning electron microscopy (SEM, Nova NanoSEM 200, FEI). To determine cytotoxicity of CNTs in contact with macrophages, ToxiLight®BioAssay Kit and ViaLight® Plus Kit tests (LONZA Rockland, Inc.) were used. The ViaLight® Plus Kit is intended for rapid and safe detection of proliferation and cytotoxicity of mammalian cells and cell lines in culture by determination of their adenosine triphosphate (ATP) levels. The ToxiLight® BioAssay is a non-destructive bioluminescent cytotoxicity assay designed to measure toxicity in mammalian cells and cell lines in culture. The kit quantitatively measures the release of adenylate kinase (AK) from damaged cells. The cytotoxicity was measured on the third day after seeding.
The results were expressed as mean ± SD obtained from 8 to 11 samples for each experimental group. Significant effects (p < 0.05) were determined using the unpaired Student’s t test.
Results and discussion
A strong oxidative environment of acid mixtures has an impact on CNT purity and chemistry. During synthesis of CNTs using both chemical vapour deposition (CVD) and electrical discharge, transition metal catalysts such as Fe, Co, Ni, Mo, etc. are often used. The exceptional ability of transition metals to catalyse CNT formation is primarily linked to their catalytic activity for the decomposition of carbon compounds, their ability to form carbides and the possibility for carbon to diffuse through and over the metals extremely rapidly (Sinnott et al. 1999; Dupuis 2005).
Metal content in the CNTs
Metal content (wt%)
Metals, such as Fe, Ni and Cu, are know to induce the formation of ROS through a Fenton-like reaction and induce intracellular oxidative stress either in a direct or indirect way via extracellular pathways (Kagan et al. 2006; Pulskamp et al. 2007).
The oxidative treatment of CNTs with the mixture of H2SO4 and HNO3 is a useful technique for removal of impurities and simultaneously to incorporate chemical groups on their surface. All types of nanotubes were characterised by Fourier transform infrared (FT-IR) spectroscopy to identify chemical groups.
The FT-IR absorption spectrum of MWCNT-NH shows a considerably attenuated band at 1,710 cm−1. This probably indicates the change of chemical circumstance of C=O in the carboxyl groups due to amino bond formation. It can be observed that the peak of νC=O was moved at 1,653 cm−1 for MWCNT-NH. Ethylenediamine functionalisation of MWCNT-F was further evidenced by the characteristic amine peaks. The presence of the band especially at 1,566 cm−1 is the most important one to characterise the chemical state of N for amine and should be ascribed to δN–H. Moreover, the C–N stretching vibration peak of ethylenediamine was observed at 1,094 cm−1 for MWCNT-NH. The broad band at 3,300–3,600 cm−1 was due to the NH2 stretch of the amine group. The FT-IR results verify that amines were covalently attached to the MWCNTs (Chen et al. 1998). The presence of the chemical group on CNT’s surface confirms the effectiveness of functionalisation methods.
XPS spectrum of the MWCNTs after purification (MWCNT-F) and functionalisation process (MWCNT-NH)
Atomic ratio (%)
O/C atomic ratio
The MWCNT after chemical purification in the acid mixture (MWCNT-F) contained three oxygen-containing bonds (O1S) (Fig. 4b). The peak at 530.6 eV is completely removed from the sample in comparison with as-prepared MWCNT. The absence of this peak indicates that the metal catalyst residue is removed from the sample. After chemical oxidation, the concentration of metal catalysts is significantly reduced in CNTs, which was also confirmed by the ICP method. Moreover, a significant difference between MWCNT and MWCNT-F was observed due to the presence of the peak located at around 532.9 eV. The oxygen atomic content for this peak deduced from XPS analysis of MWCNT-F was almost twice as high (42.30 %) as compared to MWCNT (22.21 %), which confirms the effectiveness of functionalisation methods of their surfaces. Additionally, a higher nitrogen content is observed for nanotubes after functionalisation in ethylenediamine (MWCNT-NH) in comparison with MWCNT and MWCNT-F (Table 1). The increasing content of nitrogen (N1S) with the decreasing oxygen concentration (O1S) for MWCNT-NH in comparison with MWCNT-F suggested the presence of amine groups on the surface of CNTs. The analysis of a broad C1S peak shows that its component at around 289 eV attributed to sp2 carbon bound to two oxygen atoms (O–C=O) both for MWCNT-F and MWCNT-NH are different. Decreasing the relative concentration of carboxylate acid groups for MWCNT-NH (5.20 %) in comparison with MWCNT-F (7.17 %) might suggest that they are converted to amine groups.
Morphology and structure of CNTs
The differences in morphology of MWCNTs before and after acid oxidation were observed. The observed changes in MWCNT-Fs in comparison to the as-prepared MWCNT are connected with decreases in length.
Raman spectroscopy is a sensitive technique for characterisation of carbon materials, including molecular allotropes of carbon. The Raman spectra of carbon nanoforms enable the differences in their structural features to be distinguished, and also those between the fullerenes and tubular structures referring to CNTs.
Size distribution of CNTs
Figure 9 compares the agglomerate size distributions of CNTs in water suspension preparations through sonication with ultrasound, for 15 min, using PALMER INSTRUMENTS (Model: CP 130 PB, 130 W power, 20 kHz). Bimodal distribution of MWCNT before oxidation was observed (mean values: 270 and 900 nm, respectively) (Fig. 9). The size distribution of MWCNT-F (after oxidation) was completely different in comparison with pristine MWCNT (Fig. 9). These results confirm the influence of the chemical treatment on the dispersion of carbon aggregates in water. The size distribution of MWCNT-F is shifted towards lower values. The mean sizes of MWCNT-F were 29 and 124 nm, respectively. The presence of carboxyl groups on the CNT surface change their wettability from hydrophobic to hydrophilic, which is demonstrated by facilitating the dispersion of nanotubes in an aqueous solution. Another very important observation after oxidising treatment is the reduction of the length of the CNTs. The size distribution of MWCNT-NH is different in comparison to oxidised MWCNT (MWCNT-F), though a more similar to as-prepared MWCNT. The mean sizes of MWCNT-NH are 148 and 553 nm, respectively. After functionalisation of MWCNT-F in ethylenediamnie the C=O bonds of the carboxyl groups are replaced by amine group resulting in the surface chemistry changes of nanotubes. Therefore, the nanotubes lose their affinity to the aqueous solution, resulting in poor dispersity and a stronger tendency to re-agglomeration.
In vitro investigation of CNTs
For assessment of cytotoxicity and cell transport of different types of CNTs, the macrophage cell line (RAW 264.7) was used. Before cells were exposed to the CNTs, all types of samples (MWCNT, MWCNT-F and MWCNT-NH) were immersed in PBS and dispersed for 2 min using a tip sonicator (PALMER INSTRUMENTS, Model: CP 130 PB). From each of the solutions containing MWCNT, MWCNT-F and MWCNT-NH, equal portions of these materials were transferred to cells in culture medium. Two concentrations of CNTs, i.e. 38 and 80 μg/ml were used for experiments.
Analysing the impact of the nanotube concentration on cell proliferation revealed the significant difference for samples of both oxidised (MWCNT-F) and functionalised (MWCNT-NH). For both the samples, cell proliferation decreased with increasing concentration of CNTs. The results may suggest that MWCNT-Fs have a predominantly adverse impact on cell response. The physicochemical traits of these samples such as dimension, surface chemistry, dispersal and behaviour in cell culture observed both under optical and SEM may help to explain their effects on cell proliferation. MWCNT-F interact with the cells much more easily than the other samples by two mechanisms, namely, endocytosis/phagocytosis and an energy-independent passive process. Although the MWCNT-F sample produces the greatest reduction in cell proliferation of the macrophages compared to the control condition (Fig. 18a), according to the ToxiLight and ViaLight tests, it does not exhibit the highest toxicity compared to control (Fig. 18b). The results obtained from this test show that the cell viability for this sample is not lower than that obtained for MWCNT and MWCNT-NH. For lower concentrations of MWCNT-F (38 μg/ml), the cell viability is higher in comparison to MWCNTs and MWCNT-NH. This indicates that the mechanism of cell growth in contact with MWCNT-F differs as compared to the other samples. The higher number of cells in direct contact with this kind of nanotube than that observed for MWCNTs and MWCNT-NHs is due to a better dispersion of these materials in culture medium. A high concentration of MWCNT-F in macrophages may decrease the cell proliferation process by interfering with the mitotic apparatus without negative consequences on cell viability.
Surprisingly, although the as-prepared MWCNTs produced the least reduction in cell proliferation with reference to control, the viability of cells exposed to this sample was substantially reduced with respect to control (Fig. 18b). A possible explanation is the presence of MWCNT’s agglomerates, which are surrounded by numerous cells attempting to phagocytose them (Fig. 12a). When phagocytes meet particles that are too large to be phagocytosed, a phenomenon called frustrated phagocytosis may occur, in which proteolytic enzymes and toxic substances are released out from the cell (Brown et al. 2007; Anderson et al. 2008; Sanchez et al. 2011). These substances can damage the surrounding cells. Another reason related to a decrease in the cell viability for MWCNT is their length. High aspect ratio nanoparticles can induce frustrated phagocytosis and formation of multi-nucleated giant cells similar to the response of macrophages to asbestos fibers or CNTs following pharyngeal aspiration or interaperitoneal or pleural injection (Shvedova et al. 2012; Cheng et al. 2009; Sanchez et al. 2011; Porter et al. 2010; Donaldson et al. 2010; Mercer et al. 2010).
According to our previous works and the results presented by other authors the influence of metal residuals (catalysts used for CNT manufacture) on in vitro cellular response is significantly lower than effect of the length of the nanotubes and their agglomerated form (Poland et al. 2008; Fraczek et al. 2008; Sanchez et al. 2011; Zhao and Liu 2012). Impurities, including metallic catalyst particles within the CNTs, have been suggested to serve as a catalyst for oxidative stress. Negative influence of metallic catalysts on cells response is mainly observed for iron (Kagan et al. 2006; Guo et al. 2007; Hiraishi et al. 1991). Nickel was found to have harmful impact on the cells response; established human carcinogen that induces gene silencing and hypoxia signalling through mechanisms involving intracellular nickel cation (Liu et al. 2007). However, the influence of metal catalyst on cells response depends significantly on its concentration. In our samples, nickel content is 1.2 wt% for MWCNT and 0.1 wt% for MWCNT-F, i.e. its concentration is relatively low. Moreover, a part of the metal residue is entirely covered with CNT carbon graphene layers, which may also prevent its negative effect on the cellular response. Thus, metal residue in our samples has a negligible effect on the early cell response. A more significant effect of metal residue may be expected after a longer time of contact of CNT with cells culture. Thus, major factors affecting cells proliferation and their viability after 3-day culture are the presence of agglomerated forms of nanotubes and their length. It cannot be excluded, however, that negative effect of the metallic residues will reveal after a longer contact time with cells.
The cell viability level in contact with CNTs functionalised in ethylenediamine (MWCNT-NH) is between the viability of MWCNT and MWCNT-F. This result indicates that the presence of amine groups has no significant influence on cell behaviour at a low concentration of CNTs (38 μg/ml). In this case, the most important factor in cellular response is the dispersion of CNTs.
However, the concentration of CNTs is not without significance on cell viability. The decrease in cell viability with increase in CNT concentration (80 μg/ml) is especially visible for nanotubes after oxidation (MWCNT-F) and functionalisation (MWCNT-NH) processes.
Three types of MWCNTs were investigated in this study. As-prepared CNTs (MWCNTs) were purified using concentrated acids (MWCNT-F) and then functionalised using ethylenediamine (MWCNT-NH). The results revealed that the same materials (MWCNTs) treated chemically may have a different influence on macrophage response. The importance of knowledge was proved concerning the physicochemical properties of the material to interpret the results of biological research. The treatment of CNTs in acidic medium affects their purity and introduces carboxyl groups on their surface. This process can also lead to changes in the dimensions of nanotubes. These factors in turn have a significant impact on their behaviour in culture media and consequently on the cellular response. A shorter and more hydrophilic MWCNT-F is more easily transferred inside the cells, probably through the phagocytosis process and free passage by cell membrane as compared to as-prepared MWCNTs and nanotubes in the form of agglomerates. The presence of CNTs inside the cells changes their morphology and slows down the processes of cell proliferation. At higher concentrations (up to 80 μg/ml) of this type of nanotubes, an adverse effect on cell viability was observed. Similar behaviour was observed for the nanotubes containing amino groups (MWCNT-NH). The presence of CNTs agglomerates distinctly decreases cell viability as a result of frustrated phagocytosis.
This work has been supported by the Marie Curie Actions— Industry-Academia Partnerships and Pathways (IAPP), FP7- PEOPLE-IAPP-2008, project number 230766.
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