Carboxylation of multiwalled carbon nanotubes reduces their toxicity in primary human alveolar macrophages

Sinbad Sweeney a, Sheng Hu b, Pakatip Ruenraroengsak ac, Shu Chen c, Andrew Gow d, Stephan Schwander e, Junfeng (Jim) Zhang f, Kian Fan Chung g, Mary P. Ryan b, Alexandra E. Porter c, Milo S. Shaffer b and Teresa D. Tetley *a
aLung Cell Biology, Airways Disease, National Heart & Lung Institute, Imperial College London, London, UK. E-mail: t.tetley@imperial.ac.uk
bDepartment of Chemistry and London Centre for Nanotechnology, Imperial College London, London, UK
cDepartment of Materials and London Centre for Nanotechnology, Imperial College London, London, UK
dDepartment of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey, USA
eDepartment of Environmental and Occupational Health, University of Medicine and Dentistry, School of Public Health, Rutgers University, New Jersey, USA
fDivision of Environmental Sciences & Policy, Nicholas School of the Environment, Duke University, Durham, USA
gRespiratory Medicine and Experimental Studies Unit Airways Disease, National Heart & Lung Institute, Imperial College London, London, UK

Received 1st March 2016 , Accepted 9th September 2016

First published on 26th September 2016


Surface functionalisation of multiwalled carbon nanotubes (MWCNT) is commonly used to facilitate their various and diverse applications. Inhaled nanomaterials, such as MWCNTs, have a high deposition rate in the alveolar units of the deep lung, where alveolar macrophages (AM) provide the front line of cellular immune defence by removing foreign matter (microbes, particles etc.). The toxicity of MWCNTs (with or without functionalisation) towards primary human AMs is not known. We investigated the physicochemical characteristics and toxicity of two MWCNT materials: acid purified ‘Purified-MWCNT’ and concentrated acid functionalised ‘COOH-MWCNT’. We hypothesised that the bioreactivity with primary human AM would differ between the materials. Full characterisation of the MWCNTs revealed that –COOH functionalisation yielded shorter MWCNTs, accompanied by a greater occurrence of framework defects, in comparison to Purified-MWCNT. In agreement with our hypothesis that the bioreactivity would differ, Purified-MWCNT were significantly more toxic as measured by reduced cell viability and increased inflammatory mediator release. For example, IL-1β and IL-8 release by AMs significantly increased 3.5- and 2.4-fold, respectively (P < 0.05), 24 hours after treatment with Purified-MWCNT. In contrast, IL-1β and IL-8 release by AMs did not significantly change 24 hours after treatment with COOH-MWCNT. We determined that the mechanism of this toxicity is likely due to activation of the inflammasome, as lipopolysaccharide priming of primary human AMs was necessary to see the inflammatory response and this was accompanied by lysosomal disruption and increased generation of reactive oxygen species. This study contributes further to our understanding of the effects of MWCNTs and surface modification on highly relevant human lung AMs; the findings have important implications for the manufacture, application and use of MWCNTs. In particular, this is relevant where applications prefer biocompatible MWCNTs.



Nano impact

Surface functionalisation of multiwalled carbon nanotubes (MWCNT) is commonly used to facilitate their various and diverse applications. Inhaled nanomaterials, such as MWCNTs, have a high deposition rate in the alveolar units of the deep lung. The toxicity of MWCNTs towards primary human AMs is not known and potentially toxic and biopersistant materials may be entering the environment. Purified-MWCNTs (opposed to –COOH) were significantly more toxic as measured by reduced cell viability and increased inflammatory mediator release. We determined that the mechanism of this toxicity is likely due to activation of the inflammasome. This study contributes further to our understanding of the effects of MWCNTs and surface modification on highly relevant human lung AMs; the findings have important implications for the manufacture, application and use of MWCNTs.

Introduction

Research and commercial development of carbon nanotubes (CNT) continues apace, with applications in the electronics,1 energy storage,2 automotive, aerospace3 and biotechnology (including medical devices and drug delivery)4 sectors. CNTs are cylinders of one or more layers of graphene, denoted single-walled (SWCNT) or multi-walled (MWCNT), respectively. Surface functionalisation of MWCNTs is commonly used to facilitate their various and diverse applications. For example, hydroxyl (–OH) functionalisation has been shown to improve the mechanical properties of polymer/CNT nanocomposites,5 while carboxyl functionalisation (–COOH) has been used to facilitate aqueous dispersion of MWCNTs and the attachment of drug molecules to their surface, for therapeutic delivery.6 Acid purification is one of the most commonly used methods to remove metal impurities (catalysts) from chemical vapour deposition-grown MWCNTs, and it is generally achieved by treating MWCNTs with low concentrations of acid solution, such as 1 M HNO3. Treatments using strong acids, particularly mixtures of H2SO4 and HNO3, are also frequently used in order to covalently functionalise the MWCNTs framework with –COOH groups which are suitable for further modification and allow carboxyl functionalised MWCNTs to be dispersed in aqueous solutions for those applications previously described.

Human exposure to MWCNTs may occur at any stage of the material's life cycle. A number of potential routes of exposure are possible, however inhalation of MWCNTs is a primary concern. Furthermore, it is generally accepted that there is a high rate of deposition of inhaled nano-sized particles in the alveolar region of the lung.7–9 It should be emphasised that even though MWCNTs may have lengths extending into the micron range, it is the nano-sized diameter of MWCNTs that will likely determine their alveolar deposition i.e. as seen with inhaled fibres, where their longitudinal axis that aligns with the airstream.10

Alveolar macrophages (AM) provide the front line of cellular immune defence by removing foreign matter (microbes, particles etc.) from the alveolar space. MWCNT functionalisation has been shown to be an important determinant of toxicity in various rodent macrophages (cell lines and primary macrophage cells) and in human monocyte-derived macrophages. For example, using the RAW 264.7 murine macrophage cell line, Zhang et al. showed that purified and polyethylene glycol or COOH-functionalised MWCNTs induced cytotoxicity while the COOH-functionalised material also induced significant inflammatory responses.11 Contrastingly, Fraczek-Szczypta et al. showed that COOH functionalisation of MWCNTs decreased their toxicity in murine macrophages RAW 264.7, compared to equivalent but unfunctionalised MWCNTs.12 Using the human monocyte cell line, U937 (differentiated to a macrophage-phenotype), Zhu et al. demonstrated that COOH-functionalised MWCNTs resulted in greater cellular uptake when compared to unfunctionalised MWCNTs. The authors also showed that COOH-functionalised MWCNTs induced a greater reduction in U937 viability when compared to unfunctionalised MWCNTs.13 Conversely, Gasser et al. showed that COOH functionalisation has a negligible effect on the response of human monocyte-derived macrophages (in terms of cell viability, reactive oxygen species generation and inflammatory mediator release) when compared to unfunctionalised MWCNTs.14

However, the effect of MWCNT functionalisation on the material's toxicity in primary human AMs remains unstudied. COOH functionalisation of MWCNT is one of the most commonly performed surface modifications. We have characterised two typical types of MWCNTs, acid-treated to differing degrees to produce purified and carboxlic acid functionalised forms and provide comprehensive comparisons of these materials, measuring a range of physicochemical properties. The purified and –COOH functionalised samples are referred to as Purified-MWCNTs and COOH-MWCNTs, respectively. Further, we examined the comparative toxicity of these MWCNTs towards freshly isolated primary human AMs, as one of the first cellular targets of particulate material within the alveolar respiratory unit. Based on current literature, we hypothesised that the two MWCNT materials would yield a differential toxicity profile in primary human AMs.

Methods

MWCNT samples

Unfunctionalised MWCNTs were manufactured by and purchased from CheapTubes, Inc. Carboxyl functionalisation of these MWCNTs was performed by Professor Som Mitra, New Jersey Institute of Technology, yielding the material denoted herein ‘COOH-MWCNT’, by reaction with 1[thin space (1/6-em)]:[thin space (1/6-em)]1 concentrated H2SO4 and HNO3. According to Prof Mitra, the reaction was carried out at a pre-set temperature of 140 °C for 20 min. After cooling to room temperature, the product was vacuum filtered, pore size 10 μm using deionized water, until the filtrate reached a neutral pH. COOH-MWCNTs were then dried in a vacuum oven at 70 °C until a constant weight was reached. Separately, purified MWCNTs, denoted herein as ‘Purified-MWCNT’, were prepared by washing the as-received MWCNT with 1 M HNO3. According to Prof Mitra, the reaction was carried out at a pre-set temperature of 100 °C for 10 min. After cooling to room temperature, the product was vacuum filtered (10 μm pore size) and washed using deionized (DI) water, until the permeate reached a neutral pH. Purified-MWCNTs were then dried in a vacuum oven at 65 °C until samples reached a constant weight. Neither Purified-MWCNT nor COOH-MWCNT were received as guaranteed endotoxin-free. We therefore suspended Purified-MWCNT and COOH-MWCNT in Milli-Q ultrapure water, gently sonicated them in a sonicating water bath (135 W, 42 kHz; VWR) for 1 hour (in 10 minute intervals, with vortexing and cooling periods between cycles). Following sonication, the MWCNTs were pelleted by centrifugation at 17[thin space (1/6-em)]000g, the supernatant (containing any removed endotoxin) was discarded and the MWCNT pellet was washed three times with repeated rounds of resuspension in Milli-Q ultrapure water and centrifugation at 17[thin space (1/6-em)]000g. Both Purified-MWCNT and COOH-MWCNT were then assayed for endotoxin contamination, using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, UK) and both were below the 0.1 EU mL−1 limit of detection.

MWCNT characterisation

Scanning electron microscopy. CNT samples were dispersed in ethanol using 5 min water bath sonication (45 kHz, 80 W, VWR International, USA). One droplet of the dilute CNT dispersion was deposited onto a pure aluminium stub and the sample was then imaged by scanning electron microscopy (SEM). SEM images were obtained on a GEMINI LEO 1525 FEGSEM at an accelerating voltage of 6 kV. The lengths of CNTs were measured from the SEM images by using an image analysis program (ImageJ, NIST, US); more than 100 CNTs were measured.
Thermogravimetric analysis. Thermogravimetric analysis (TGA) analyses were carried out using a Mettler TGA/DSC 1, by heating 5.0 ± 0.5 mg MWCNT samples to 100 °C, under a N2 atmosphere (50 mL min−1), and holding isothermally for 15 minutes to remove residual water and/or solvent; the temperature was then increased from 100 °C to 850 °C at a constant ramping rate of 10 °C min−1 under flowing N2 or air (50 mL min−1).
Elemental analysis. A qualitative and quantitative estimation of metal impurities in the MWCNT samples was performed using energy dispersive X-ray spectroscopy (EDS) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. SEM images and EDS spectra were collected using a Hitachi S3000 (Hitachi Group) equipped with a tungsten filament electron source. Five spectra in five different areas where the copper tape was completely covered were collected for each sample for 30 seconds, each with approximately 30% dead time. The background was subtracted and the spectra were analysed for a qualitative guidance of elemental composition. For ICP-MS analysis, an Agilent ICP-MS 7500CX equipped with micro-mist nebulizer, standard sample introduction system, and integrated auto-sampler, operated in He mode in Agilent's proprietary ORS (Octopole Reaction System) was used. Calibration standards were prepared using NIST SRM-3162A titanium standard solution, NIST SRM-3136 nickel standard solution, NIST SRM-3126A iron standard solution, and NIST SRM-3168A zinc standard solution. Internal standards were prepared using NIST SRM-3121 gold standard solution and NIST SRM-3140 platinum standard solution. All samples were digested using trace element grade concentrated (70%) nitric acid (HNO3).
Specific surface area. The measurements of adsorption and desorption isotherms of nitrogen at 77 K were carried out on approximately 100 mg MWCNTs using a Micromeritics ASAP 2010 apparatus. Specific surface areas were calculated according to the Brunauer, Emmet and Teller (BET) equation from the adsorption isotherm in the relative pressure range of 0.05–0.20 p/p0. The distributions and total volumes of mesopores were calculated according to Barret, Joyner and Halenda (BJH-method) from the desorption branch of the isotherm. The total micropore volumes were determined from the adsorption branch of the isotherm using the t-plot method.

Ethics statement

The human lung tissue used in this study was surplus tissue obtained following resection for lung carcinoma from subjects without any known co-morbidities. Written informed consent was obtained for all samples and the study was carried out with the approval of the Royal Brompton and Harefield Ethical Committee (Ref: 08/H0708/73).

AM cell model

Primary human AMs were isolated from human lung of grossly normal appearance obtained following resection for lung carcinoma, as described previously.15 AMs were isolated from a minimum of five subjects per experiment. Briefly, lung tissue sections were perfused by injection of 0.15 M sterile sodium chloride saline solution until the draining lavage ran clear and the cell count was <1 × 104 cells per ml. The saline perfusate was collected and centrifuged at 240 × g for 10 minutes at 20 °C. The cell pellet was re-suspended in serum-free RPMI cell culture medium containing 1% penicillin/streptomycin/L-glutamine (PSG) and plated for respective experiments. After AM adherence during incubation at 37 °C with 5% CO2 (approximately three hours), culture medium was removed and non-adherent cells (including contaminating blood) were removed by gentle PBS washing. For experiments, AM cultures were maintained in serum-free RPMI cell culture medium containing 1% PSG, incubated at 37 °C with 5% CO2.

AM treatment with MWCNTs

AMs were treated with 25 μg ml−1 (prepared in RPMI 1640 culture medium) of Purified-MWCNT or COOH-MWCNT for 4 and 24 hours. Both MWCNT preparations were sonicated in a sonicating water bath (135 W, 42 kHz; VWR) for 30 seconds prior to cell culture exposure. A 25 μg ml−1 dose was chosen because preliminary studies by us revealed it induced the greatest magnitude of inflammatory mediator release (from a pilot study of LPS-primed AMs examining concentration range of 1–50 μg ml−1 Purified-MWCNT; see Fig. S5), thus it would give us the best opportunity of seeing any differences in bioreactivity between the two MWCNT materials).

MWCNT uptake analysis using transmission electron microscopy

Following the MWCNT exposure AMs were fixed with 2.5% glutaraldehyde for 2 h and they were rinsed with 2.5% glutaraldehyde in 0.1 M HEPES pH 7.2 for 2 h and then rinsed with 0.1 M HEPES pH 7.2 (×3). Samples were next post-fixed in 1% osmium tetroxide for 30 min and dehydrated using a graded series of ethanol (50%, 70%, 90% and dry 100% ethanol) for 15 min (×3) in each solution. Samples were incubated with 25, 50, 75 and 100% in ethanol of Araldite® for 20 min (×3) each and the fresh 100% Araldite® was added and incubated at 60 °C for 24 h. The embedded samples were sectioned with a diamond knife (DiATOME) to a thickness of 70–120 nm.

The sections were observed in a JEOL 2000 transmission electron microscope (TEM, JEOL 2000FX, Japan) operated at an accelerating voltage of 80 kV to increase contrast from cell organelles. The uptake analysis was carried out as described previously.16 Three sections from three different embedded capsules (n = 3) were viewed per sample; 30 cells were surveyed in each section (90 cells were surveyed in total). The percentage of AM MWCNT internalisation was calculated in proportion to the total cell count and data were presented as mean ± SD (n = 3).

High resolution TEM

MWNTs were dispersed in ethanol by bath sonication for 30 seconds. Single drop of MWNTs suspension was drop-casted on holey carbon copper TEM grids (TAAB), and dried under vacuum. High-resolution TEM (HRTEM) imaging was carried out on a Cs-aberration-corrected FEI Titan 80/300 with an accelerating voltage of 80 kV.

AM viability

MTS is a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium that can be reduced by dehydrogenase enzymes within living cells demonstrating cellular metabolic activity and cell viability. Such reduction in the presence of an electron-coupling reagent, phenazine ethosulfate (MPS), results in the production of formazan product that is soluble in tissue culture medium and the absorbance of this formazan product can be measured using spectrophotometry. AMs were plated on 96-well plates at density of 150[thin space (1/6-em)]000 cells per well in RPMI 1640 culture medium (containing 5% L-glutamine–penicillin–streptomycin) at 37 °C with 5% CO2. Four and twenty-four hours after treatment with MWCNTs, AMs were rinsed with PBS and incubated with RPMI 1640 culture medium containing the MTS reagent according to the manufacturer's protocol for 1.5 h (CellTiter 96® AQueous One Solution Assay, Promega, USA). The absorbance of formazan product in the culture medium was then read using a spectrophotometer at a wavelength of 490 nm. We have established that the MWCNTs used in this study do not interfere with the measurement of formazan product and therefore do not interfere with the MTS cell viability assay (data not shown).

Measurement of inflammatory mediator release

AMs were studied with and without lipopolysaccharide (LPS) priming. LPS-primed AMs were treated with 10 ng of LPS (Enzo Life Sciences) overnight prior to MWCNT treatment. Four and twenty-four hours post-treatment with Purified-MWCNT or COOH-MWCNT, AM mediator-conditioned medium was assayed for concentrations of the inflammatory mediators interleukin-8 (human IL-8) and interleukin-1 beta (human IL-1β) using sandwich enzyme-linked immunosorbent assays (ELISA). The assays were performed using DuoSet® antibody kits and according to the manufacturer's directions (R&D systems, USA). In the ELISA assay, we assessed potential MWCNT interference by spiking a 1000 pg ml−1 standard solution of IL-8 or IL-1β with 25 μg ml−1 of each MWCNT material. ELISA was performed on the standard alone and the MWCNT spiked standard. We did not observe any significant differences in the measured concentration of the non-spiked and MWCNT spiked standard, and therefore concluded that the MWCNTs used in this study did not interfere with the ELISA assay.

Reactive oxygen species generation

Four and twenty-four hours post-treatment with MWCNTs, intracellular AM reactive oxygen species (ROS; key contributors to cellular oxidative stress) were detected by measuring the oxidation of florescent dihydroethidium (DHE). DHE is readily permeable to cells and is oxidized by ROS, primarily superoxide, to yield ethidium. Ethidium subsequently binds to DNA, which produces a detectable red fluorescence. In the present study, ROS production was measured with the DHE probe using a variation of the protocol derived from Castilho et al.17 AMs were plated on 96-well plates (black, with a clear bottom) at a density of 150[thin space (1/6-em)]000 cells per well in RPMI 1640 culture medium (containing 5% L-glutamine–penicillin–streptomycin) at 37 °C with 5% CO2. One and five days post-treatment with MWCNTs, AMs were rinsed with PBS and incubated with RPMI 1640 culture medium containing 100 μl of 10 μM DHE (Invitrogen, Paisley UK) in RPMI 1640 culture medium for 30 minutes. At the end of DHE incubation, AMs were washed twice to remove extracellular probe and read in a fluorescence plate reader at an EX/EM of 485/595 nm. We assessed potential MWCNT interference in the DHE assay by adding the DHE probe to each of the MWCNT materials (using a 25 μg ml−1 MWCNT dose) in a cell-free setup. We did not observe any spontaneous ethidium production with detectable red fluorescence, and therefore concluded that the MWCNTs used in this study did not interfere with the DHE assay.

Lysosomal integrity

The lysosomal probe, Lysotracker Red DND-99 (Invitrogen-Molecular Probes), was used to assess the number of lysosomes whose integrity remained intact following 4 and 24 hour treatment with Purified-MWCNT or COOH-MWCNT. Briefly, AMs were plated on 96-well plates (black, with a clear bottom) at a density of 150[thin space (1/6-em)]000 cells per well in RPMI 1640 culture medium (containing 5% L-glutamine–penicillin–streptomycin) at 37 °C with 5% CO2. Four and 24 hours post-treatment with MWCNTs, AMs were rinsed with PBS and incubated with RPMI 1640 culture medium containing 50 nM Lysotracker Red DND-99 probe, for 40 minutes. At the end of the incubation period, AMs were washed twice to remove residual probe and AMs were then imaged using a fluorescent microscope and red fluorescence intensity was quantified using a fluorescence plate reader at Ex/Em: 485/595 nm (Tecan, Switzerland).

Statistical analyses

Data from AM viability, inflammatory mediator release, ROS generation and lysosomal integrity experiments are presented as the mean ± standard error where six independent experiments were performed using six separate donor tissues for AM harvest. Significant differences between non-treated and MWCNT-treated AMs were determined using non-parametric Mann–Whitney test. In all analyses, a P value <0.05 was considered significant.

Results

MWCNT characterisation

Due to the difference in preparation protocols (mainly acid concentration and reaction temperature), the physicochemical properties of purified MWCNTs and acid oxidised MWCNTs (COOH-MWCNTs) are expected to be significantly different, in terms of the oxygen contents, surface functional groups, surface area etc., as shown by the various characterisations.
SEM determination of length distribution. Length distributions of the MWCNTs following acid purification and acid functionalisation treatments, obtained from over 100 MWCNTs imaged on independent positions across the specimen,18 are shown in Table 1 (the histogram of length distribution can be referred to in Fig. S1). After mild acid treatment, Purified-MWCNTs showed a length distribution in the range of 0.2 μm to 2 μm with an average length of 1.1 μm, which was not remotely different from as-received MWCNTs with nominal length ranging from 0.5 to 2 μm (as stated by the manufacturer). However, the acid functionalised COOH-MWCNTs were significantly shortened, with a reduced average length of 652 nm, attributed to cutting and/or end etching during functionalisation, as described by us previously.19
Table 1 Nominal and characterised MWCNT length distribution
Sample Length (nm, mean ± SE, SD)
SE: standard error. SD: standard deviation.
Purified-MWCNTs 1122 ± 44, 495
COOH-MWCNTs 652 ± 28, 327


Thermal gravimetric analysis (TGA). The two types of MWCNTs showed apparently different decomposition behaviours as determined by TGA under N2 atmosphere (Fig. 1). It is worth mentioning that the moisture within each sample was removed during the isothermal treatment at 100 °C (refer to Fig. 1 and Fig. S2), which accounted approximately 3.5 wt% and 1.5 wt% for COOH- and purified-MWCNTs, respectively, as the more oxidised MWCNTs are more hydrophilic. It is widely reported that the COOH functionalities decompose in the temperature range of 180–400 °C.20 Furthermore, it's been know that the concentrated acid oxidation reaction will produce many other types of surface oxygen-containing groups apart from carboxylic acid groups, including hydroxyl and lactone groups,21,22 which are thermally stable up to approximately 800 °C.20 Therefore, the weight loss shown in the TGA curves are collectively contributed by various surface functional groups, together with carbonaceous debris generated during acid treatment.21 While it is difficult to accurately quantify the COOH on the CNT surface, the TGA indicated a significantly increased concentration of functionalities for COOH-MWCNTs. The carbon framework, on the other hand, is expected to be stable under the current TGA measurement conditions, in inert atmosphere, which contributed to the residue weight (refer to the table inserted in Fig. 1).
image file: c6en00055j-f1.tif
Fig. 1 TGA profile (100–850 °C) of Purified- and COOH-functionalised MWCNTs, measurements were carried out under N2 atmosphere.
Elemental analysis. The ICP-MS, SEM-EDS and STEM-EDS determined elemental content of carbon, oxygen, and metal impurities from the two MWCNT samples are summarised in Table 2 and Table S1. The metal impurities included residue amounts of Ni, Fe, Ti and Zn, and the functionalisation reaction with more intense acid treatment removed a significant amount of the metal, apart from Zn which accounted for the least amount among the series. Increases in weight percentage of oxygen were observed in the COOH-MWCNT as compared to Purified-MWCNT, potentially indicating the presence of additional oxygen on the surface. However, it is worth mentioning that neither the SEM nor the STEM EDS results were quantitative measurements (refer to ESI).
Table 2 Elemental analysis by ICP-MS
Sample ICP-MS
(μg of element per gram of sample)
Ni Fe Ti Zn
Purified-MWCNTs (4.143 ± 0.173) × 103 526.882 ± 20.015 14.22 ± 0.57 7.32 ± 0.31
COOH-MWCNTs (1.245 0.083) × 103 BLOQ 7.42 ± 0.28 39.39 ± 1.50


BET surface area. Following BET analysis (Table 3), COOH-MWCNTs showed a higher BET surface area compared to Purified-MWCNT, along with significantly increased micropore volume and micropore area. Several effects are introduced by the functionalisation process, including an increase in MWCNT surface roughness, generation of additional defects and opening/shortening effects consistent with length distribution data shown earlier.
Table 3 BET surface area analysis of MWCNT samples
Sample BET surface area (m2 g−1) Micropore volume (m3 g−1) Micropore area (m2 g−1)
Purified-MWCNT 174.2 ± 0.6 6.8 × 10−10 2.9
COOH-MWCNT 209.3 ± 0.3 3.3 × 10−9 10.1


High-resolution transmission electron microscopy (HRTEM). HRTEM imaging provided a comparison of surface morphology and crystallinity between acid-functionalised and acid-purified MWCNTs (Fig. 2). A variety of defects appearing as peeling, pitting and delamination were found on both types of MWNTs. However, after treatment with concentrated acid, COOH-MWCNTs exhibit more obvious damage, with additional peeling or etching of the carbon framework. For an overview of the Purified-MWCNT and COOH-MWCNT samples, see Fig. S9.
image file: c6en00055j-f2.tif
Fig. 2 HRTEM images showing of the morphology and crystallinity of a) acid-purified Purified-MWCNTs and b) acid-functionalised COOH-MWCNTs. Circled areas showed peeling and etching of the MWCNTs framework caused by the acid functionalisation process.

In summary, the Purified-MWCNTs experience a much milder oxidation environment and are much less aggressively etched (see TEM in Fig. 2), cut (average length differs by a factor of two), and functionalised (TGA mass loss and oxygen content differ by a factor of two, Raman D/G ratio shifts significantly), compared to the COOH-MWCNTs.

MWCNT uptake by AMs. Both MWCNTs were generally observed within cell vesicles (endosome/lysosome/phago-lysosomes). The vesicular Purified-MWCNT were more tightly aggregated than the COOH-MWCNTs (Fig. 3A–D). In addition, individual MWCNTs (yellow arrows in B and D) were observed within cell vesicles, but relatively fewer single MWCNTs were present in the cytoplasm, and were mainly in the vicinity of the endosomal vesicles (black arrows, B and D). The TEM uptake analysis (Fig. 3E) showed that, at 4 h, 35% and 44% of the total AMs analysed internalised Purified-MWCNTs and COOH-MWCNT, respectively, which was significantly different (p < 0.05, n = 3 separate estimates of a total of 90 observed cells). By 24 h, this significance disappeared, and the percentage of AMs that had internalised MWCNTs rose to ∼70% (Fig. 3F). Electron micrographs of non-treated AMs can be viewed in Fig. S3.
image file: c6en00055j-f3.tif
Fig. 3 Uptake of Purified-MWCNTs (A and B) and COOH-MWCNTs (C and D), both at 25 μg ml−1, by AMs following 24 h exposure; the percentages of cells which internalised MWCNTs at 4 and 24 h are also provided (E and F, respectively) as a bar graph, mean ± SD. (A) AMs exposed to Purified-MWCNTs for 24 h with an insert image at the low magnification image. (B) is the high magnification image of the region of interest (ROI) in green square in (A). (C) AMs exposed to COOH-MWCNTs with the insert image at the low magnification image. (D) is the high magnification image of the ROI in the blue square in (C). The yellow arrows indicate the individual MWCNTs internalised within vesicles, and black arrows indicate MWCNTs penetrating the vesicular membranes (V) within AMs.
Cell viability. AM viability, four and twenty four hours after treatment with 25 μg ml−1 Purified-MWCNT or COOH-MWCNT, was measured using the MTS assay. AM viability did not significantly change 4 hours after treatment with Purified-MWCNT or COOH-MWCNT (Fig. S4). AM viability significantly decreased by 14% (P < 0.05), 24 hours after treatment with Purified-MWCNT (OD490nm = 0.53 compared to 0.62 for the untreated control), however COOH-MWCNT did not induce any significant change at this time point (OD490nm = 0.59 compared to 0.62 for the untreated control; Fig. 4 displays the normalised data).
image file: c6en00055j-f4.tif
Fig. 4 Viability of AMs after 24 hour treatment with 25 μg ml−1 Purified-MWCNT and COOH-MWCNT, as determined by the MTS assay. Cell viability data are presented as a % of the non-treated (NT) control (n = 6) ± SEM; significant differences between non-treated and treated AMs are indicated where *P < 0.05.
Inflammatory mediator release. Neither IL-1β nor IL-8 release from AMs, in the absence or presence of LPS priming, changed significantly 4 hours after treatment with 25 μg ml−1 Purified-MWCNT or COOH-MWCNT (Fig. S6). IL-1β release by AMs (LPS-primed only) significantly increased 3.5-fold (P < 0.05) 24 hours after treatment with Purified-MWCNT (Fig. 5). In contrast, IL-1β release by AMs (with or without LPS priming), did not significantly change 24 hours after treatment with COOH-MWCNT. IL-8 release by AMs (LPS-primed only) significantly increased 2.4-fold (P < 0.01) 24 hours after treatment with Purified-MWCNT (Fig. 5). In contrast, IL-8 release by AMs (with or without LPS priming), did not significantly change 24 hours after treatment with COOH-MWCNT.
image file: c6en00055j-f5.tif
Fig. 5 Inflammatory mediator release from MWCNT-treated AMs. IL-1β (A) and IL-8 (B) release from AMs (with and without LPS priming), after 24 hour treatment with Purified-MWCNT and COOH-MWCNT. Mediator release is presented as pg ml−1 (n = 6) ± SEM; significant differences between non-treated and treated AMs are indicated where *P < 0.05.
Lysosomal integrity. AM lysosomal integrity (and/or lysosome numbers) was measured four and twenty four hours after treatment with 25 μg ml−1 Purified-MWCNT or COOH-MWCNT. AM lysosomal integrity did not change significantly 4 hours after treatment with 25 μg ml−1 Purified-MWCNT or COOH-MWCNT (Fig. S7). At 24 hours, AM lysosomal integrity significantly decreased 69% (P < 0.05) after treatment with Purified-MWCNT (Fig. 6). In contrast, AM lysosomal integrity did not significantly change 24 hours after treatment with COOH-MWCNT.
image file: c6en00055j-f6.tif
Fig. 6 Lysosomal integrity (and/or lysosome numbers) of non-treated AMs and Purified-MWCNT and COOH-MWCNT treated AMs after 24 hours exposure, determined by Lysotracker probe (A; red). Graphical representation of measured fluorescence at Ex/Em 485/595 nm is also shown (B) n = 6 ± SEM; significant differences between non-treated and treated AMs are indicated where *P < 0.05; NT = non-treated.
Generation of reactive oxygen species. The fluorescent probe, DHE, was used to measure the generation of reactive oxygen species (primarily, the superoxide anion) in AMs, 4 and 24 hours after treatment with 25 μg ml−1 Purified-MWCNT or COOH-MWCNT. AM superoxide levels did not change significantly 4 hours after treatment with 25 μg ml−1 Purified-MWCNT or COOH-MWCNT (Fig. S8). At 24 hours, AM superoxide levels significantly increased 2.1-fold (P < 0.05) 24 hours after treatment with Purified-MWCNT (Fig. 7). In contrast, AM superoxide levels did not significantly change 24 hours after treatment with COOH-MWCNT.
image file: c6en00055j-f7.tif
Fig. 7 Generation of reactive oxygen species in AMs. ROS measured in AMs after 24 hour treatment with Purified-MWCNT and COOH-MWCNT, determined by the fluorescent DHE probe assay (Ex/Em 485/595); n = 6 ± SEM; significant differences between non-treated and treated AMs are indicated where *P < 0.05; NT = non-treated.

Discussion

In this study, we compared the toxicity of Purified-MWCNTs with COOH-MWCNTs using primary human AMs, the first cellular targets of inhaled particles that reach the alveolar units. The MWCNTs employed in this study were purchased from CheapTubes, who is one of the most important manufacturers and suppliers of carbon nanotubes and other carbon nanomaterials. The specific product used here is the Multi Walled Carbon Nanotubes 30–50 nm, synthesised by chemical vapour deposition (CVD) process which is the most widely used approach for mass production of CNTs.23

The study reported here is part of the very large Nano GO Consortium research centre established by NIEHS in 2010, aiming to produce comprehensive biological responses profiles of engineered nanomaterials (ENMs), including MWCNTs. The materials employed here (including the modified varients) were selected for use exactly to provide a consistent baseline for study across a large number of laboratories. They have indeed been used across several research labs whose results can be found in several other publications.24,25

The acid oxidation reaction was carried out within one of the consortium collaborators to standardize the materials. Similar products can be purchased from CheapTubes, but without the explicit detailed preparation protocols; for this reason, the consortium applied archetypical acid treatment reactions for both purification and functionalisation.

The TEM-visualised percent AM uptake of both the Purified-MWCNTs and the COOH-MWCNTs taken up by AMs was similar, particularly by 24 hours exposure, indicating that the differential effects of these MWCNTs on cellular bioreactivity depended on their physicochemistry. A measure of cell viability is the most fundamental of toxicity assays and although it does not reveal any other information on the bioreactivity of the material being applied, it does provide a useful initial assessment of differential bioreactivity. In the present study, AM viability significantly decreased by 14% (P < 0.05), 24 hours after treatment with Purified-MWCNT while COOH-MWCNT did not induce any change at this time point.

With a clear difference in the cytotoxicity of Purified-MWCNTs compared to COOH-MWCNTs (and with AMs an important component of the immune response), we were interested to evaluate the inflammatory potential of the respective MWCNT materials. AMs are constantly exposed to inhaled bacterial endotoxin (e.g. free and/or adsorbed to particulate material); under normal conditions the endotoxin level is typically not sufficient to incite a significant inflammatory response. Nevertheless, we considered that the levels might be sufficient to ‘prime’ AM activation by other exogenous agents. Therefore, in the study of the AM inflammatory response to MWCNT exposure, we also primed AM overnight with the bacterial endotoxin, LPS, prior to MWCNT exposure. The lack of inflammatory mediator release (IL-8 or IL-1β; in the absence or presence of LPS priming) after 4 hour treatment with Purified-MWCNT or COOH-MWCNT likely reflects the relatively lower uptake of the materials at this time point and/or slow induction of intracellular pathway activation. This suggests that MWCNT uptake is, at least in part, likely required for AMs to generate an inflammatory response and this is likely related to the physicochemistry of the MWCNTs themselves. The induction of the proinflammatory mediator IL-1β indicates a potential role for the inflammasome in AMs following Purified-MWCNT exposure. Indeed, the inflammasome (a multiprotein complex that regulates maturation and release of proinflammatory IL-1β and IL-18 release via caspase-1 activation)26 has been suggested as a common response mechanism in CNT-induced inflammation in vitro and in vivo. Palomaki et al. demonstrated that long, needle-like CNT and crocidolite asbestos activated secretion of IL-1β from human monocyte-derived macrophages. The authors showed that MWCNT-induced inflammasome activation depended on reactive oxygen species production, cathepsin B activity, the P2X7 receptor, and the Src and Syk tyrosine kinases.27 Similarly, Meunier et al. showed that double-walled CNTs induced IL-1β secretion from human monocytes and that this was exclusively linked to caspase-1 and Nlrp3 inflammasome activation.28 Using primary murine alveolar macrophages, Hamilton et al. showed that titanium dioxide nanowires induced inflammasome activation by lysosomal disruption.29 Thus we used the highly relevant human alveolar macrophage model to determine whether lysosomal integrity within MWCNT-exposed AMs was compromised. Strikingly, we observed that following 24 hours treatment with Purified-MWCNT, AM lysosomal membrane integrity had significantly decreased while no change was observed following treatment with COOH-MWCNT. This strongly indicates a role for lysosomal disruption in activation of the NLRP3 inflammasome in primary human alveolar macrophages, following Purified-MWCNT exposure. In combination with the ‘first signal’ provided by LPS-priming of the AMs (resulting in accumulation of intracellular pro- IL-1β), Purified-MWCNT exposure results in maturation and release of measurable IL-1β. Conversely, the absence of lysosomal disruption in AMs following COOH-MWCNT exposure would not lead to maturation of pro-IL-1β and therefore there was no induction of an inflammatory response. In agreement with observations by Hornung et al.30 and Lunov et al.,31 but in contrast to Hamilton et al.,29 we observed that the increase in lysosomal disruption following Purified-MWCNT exposure was accompanied by an increase in generation of ROS. Increased generation of ROS has also been shown to contribute to inflammasome activation32–34 and in combination with the AM lysosomal disruption seen following treatment with Purified-COOH, the pro-inflammatory mechanism in human AMs is likely related to these events.

As described by us,35 and others previously,36,37 carboxylation of MWCNTs by strong acid treatment can result in damage, such as peeling or etching of the carbon framework. Indeed, in the present study we observed that after treatment with concentrated acid, the COOH-MWCNTs showed exactly this type of damage to the carbon framework. In contrast, the structure of acid-purified Purified-MWCNTs was better maintained. It is unclear precisely how damage to the MWCNTs framework may subdue biological reactivity in this instance, however such damage may render the COOH-MWCNTs more vulnerable to degradation in lysosomes thus limiting their ability to induce lysosomal disruption. Indeed, the literature suggests that the presence of carboxyl groups in combination with associated defect sites facilitates interactions with the oxidative agents (e.g. lysosomal enzymes) promoting CNT degradation.38–40 In particular, Russier et al. demonstrated that defects in the MWCNT framework leads to increased degradation in simulated phagolysosomal fluid (a buffer used to mimic the chemical environment of phagolysosomes) and in horseradish peroxidase.41

In summary, we have demonstrated that concentrated acid –COOH functionalisation of acid-treated MWCNTs renders these materials less toxic in primary human AMs, when compared to non-functionalised Purified MWCNTs. These data contribute to our understanding of how surface modification of MWCNT might radically change their bioreactivity. These findings have important implications for the manufacture, application and use of MWCNTs. In particular, this is relevant where biodegradable MWCNTs would be an advantage, or might be considered safer, for example, in nanomedicine.

Acknowledgements

This work was funded by the NIEHS (grant number U19ES019536). The MWCNTs investigated were procured, characterized and provided by NIEHS as part of NCNHIR consortium program. We acknowledge the NCL, NCI (Frederick, MD, USA) for performing the EDS and ICP-MS elemental analysis presented in this study.

We also acknowledge Prof. Som Mitra, NJIT for preliminary characterization and carboxylation of MWCNTs. This research was also supported by the Medical Research Council and Public Health England, Centre for Environment and Health and by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. The reviews expressed in this publication are solely those of the authors and not necessarily those of the funding agency.

References

  1. W. Bauhofer and J. Z. Kovacs, Compos. Sci. Technol., 2009, 69, 1486–1498 CrossRef CAS .
  2. L. Dai, D. W. Chang, J.-B. Baek and W. Lu, Small, 2012, 8, 1130–1166 CrossRef CAS PubMed .
  3. S. R. Bakshi and A. Agarwal, Carbon, 2011, 49, 533–544 CrossRef CAS .
  4. A. J. Thorley and T. D. Tetley, Pharmacol. Ther., 2013, 140, 176–185 CrossRef CAS PubMed .
  5. L. Liu, A. H. Barber, S. Nuriel and H. D. Wagner, Adv. Funct. Mater., 2005, 15, 975–980 CrossRef CAS .
  6. M. Prato, K. Kostarelos and A. Bianco, Acc. Chem. Res., 2008, 41, 60–68 CrossRef CAS PubMed .
  7. M. Bailey, Radiat. Prot. Dosim., 1994, 53, 107–114 CAS .
  8. P. A. Jaques and C. S. Kim, Inhalation Toxicol., 2000, 12, 715–731 CrossRef CAS PubMed .
  9. C. C. Daigle, D. C. Chalupa, F. R. Gibb, P. E. Morrow, G. Oberdörster, M. J. Utell and M. W. Frampton, Inhalation Toxicol., 2003, 15, 539–552 CrossRef CAS PubMed .
  10. G. L. Kennedy and D. P. Kelly, in Fiber Toxicology, ed. D. Warheit, Acedemic Press, 1993, pp. 15–42 Search PubMed .
  11. T. Zhang, M. Tang, L. Kong, H. Li, T. Zhang, S. Zhang, Y. Xue and Y. Pu, J. Hazard. Mater., 2012, 219–220, 203–212 CrossRef CAS PubMed .
  12. A. Fraczek-Szczypta, E. Menaszek, T. B. Syeda, A. Misra, M. Alavijeh, J. Adu and S. Blazewicz, J. Nanopart. Res., 2012, 14, 1181 CrossRef PubMed .
  13. L. Zhu, A. M. Schrand, A. A. Voevodin, D. W. Chang, L. Dai and S. M. Hussain, Nanosci. Nanotechnol. Lett., 2011, 3, 88–93 CrossRef CAS .
  14. M. Gasser, P. Wick, M. J. D. Clift, F. Blank, L. Diener, B. Yan, P. Gehr, H. F. Krug and B. Rothen-Rutishauser, Part. Fibre Toxicol., 2012, 9, 17 CrossRef CAS PubMed .
  15. S. Sweeney, D. Berhanu, S. K. Misra, A. J. Thorley, E. Valsami-Jones and T. D. Tetley, Carbon, 2014, 78, 26–37 CrossRef CAS PubMed .
  16. M. Miragoli, P. Novak, P. Ruenraroengsak, A. I. Shevchuk, Y. E. Korchev, M. J. Lab, T. D. Tetley and J. Gorelik, Nanomedicine, 2013, 8, 725–737 CrossRef CAS PubMed .
  17. S. L. Budd, R. F. Castilho and D. G. Nicholls, FEBS Lett., 1997, 415, 21–24 CrossRef CAS PubMed .
  18. J. H. Lehman, M. Terrones, E. Mansfield, K. E. Hurst and V. Meunier, Carbon, 2011, 49, 2581–2602 CrossRef CAS .
  19. J. Cho, A. R. Boccaccini and M. S. P. Shaffer, Carbon, 2012, 50, 3967–3976 CrossRef CAS .
  20. G. S. Szymański, Z. Karpiński, S. Biniak and A. Świ[a with combining cedilla]tkowski, Carbon, 2002, 40, 2627–2639 CrossRef .
  21. R. Verdejo, S. Lamoriniere, B. Cottam, A. Bismarck and M. Shaffer, Chem. Commun., 2007, 513–515 RSC .
  22. Z. Wang, M. D. Shirley, S. T. Meikle, R. L. Whitby and S. V. Mikhalovsky, Carbon, 2009, 47, 73–79 CrossRef CAS .
  23. M. Kumar and Y. Ando, J. Nanosci. Nanotechnol., 2010, 10, 3739–3758 CrossRef CAS PubMed .
  24. T. Xia, N. Li and A. E. Nel, Annu. Rev. Public Health, 2009, 30, 137–150 CrossRef PubMed .
  25. J. C. Bonner, R. M. Silva, A. J. Taylor, J. M. Brown, S. C. Hilderbrand, V. Castranova, D. Porter, A. Elder, G. Oberdörster, J. R. Harkema, L. A. Bramble, T. J. Kavanagh, D. Botta, A. Nel and K. E. Pinkerton, Environ. Health Perspect., 2013, 121, 676–682 CrossRef PubMed .
  26. J. Tschopp and K. Schroder, Nat. Rev. Immunol., 2010, 10, 210–215 CrossRef CAS PubMed .
  27. J. Palomäki, E. Välimäki, J. Sund, M. Vippola, P. A. Clausen, K. A. Jensen, K. Savolainen, S. Matikainen and H. Alenius, ACS Nano, 2011, 5, 6861–6870 CrossRef PubMed .
  28. E. Meunier, A. Coste, D. Olagnier, H. Authier, L. Lefèvre, C. Dardenne, J. Bernad, M. Béraud, E. Flahaut and B. Pipy, Nanomedicine, 2012, 8, 987–995 CAS .
  29. R. F. Hamilton, N. Wu, D. Porter, M. Buford, M. Wolfarth and A. Holian, Part. Fibre Toxicol., 2009, 6, 35 CrossRef PubMed .
  30. V. Hornung, F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock, K. A. Fitzgerald and E. Latz, Nat. Immunol., 2008, 9, 847–856 CrossRef CAS PubMed .
  31. O. Lunov, T. Syrovets, C. Loos, G. U. Nienhaus, V. Mailänder, K. Landfester, M. Rouis and T. Simmet, ACS Nano, 2011, 5, 9648–9657 CrossRef CAS PubMed .
  32. F. Martinon, Eur. J. Immunol., 2010, 40, 616–619 CrossRef CAS PubMed .
  33. O. Gross, C. J. Thomas, G. Guarda and J. Tschopp, Immunol. Rev., 2011, 243, 136–151 CrossRef CAS PubMed .
  34. A. Harijith, D. L. Ebenezer and V. Natarajan, Front. Physiol., 2014, 5, 352 Search PubMed .
  35. S. Chen, S. Hu, E. F. Smith, P. Ruenraroengsak, A. J. Thorley, R. Menzel, A. E. Goode, M. P. Ryan, T. D. Tetley, A. E. Porter and M. S. P. Shaffer, Biomaterials, 2014, 35, 4729–4738 CrossRef CAS PubMed .
  36. M. N. Tchoul, W. T. Ford, G. Lolli, D. E. Resasco and S. Arepalli, Chem. Mater., 2007, 19, 5765–5772 CrossRef CAS .
  37. V. Datsyuk, M. Kalyva, K. Papagelis, J. Parthenios, D. Tasis, A. Siokou, I. Kallitsis and C. Galiotis, Carbon, 2008, 46, 833–840 CrossRef CAS .
  38. B. L. Allen, P. D. Kichambare, P. Gou, I. I. Vlasova, A. A. Kapralov, N. Konduru, V. E. Kagan and A. Star, Nano Lett., 2008, 8, 3899–3903 CrossRef CAS PubMed .
  39. B. L. Allen, G. P. Kotchey, Y. Chen, N. V. K. Yanamala, J. Klein-Seetharaman, V. E. Kagan and A. Star, J. Am. Chem. Soc., 2009, 131, 17194–17205 CrossRef CAS PubMed .
  40. V. E. Kagan, N. V. Konduru, W. Feng, B. L. Allen, J. Conroy, Y. Volkov, I. I. Vlasova, N. A. Belikova, N. Yanamala, A. Kapralov, Y. Y. Tyurina, J. Shi, E. R. Kisin, A. R. Murray, J. Franks, D. Stolz, P. Gou, J. Klein-Seetharaman, B. Fadeel, A. Star and A. A. Shvedova, Nat. Nanotechnol., 2010, 5, 354–359 CrossRef CAS PubMed .
  41. J. Russier, C. Ménard-Moyon, E. Venturelli, E. Gravel, G. Marcolongo, M. Meneghetti, E. Doris and A. Bianco, Nanoscale, 2011, 3, 893 RSC .

Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c6en00055j

This journal is © The Royal Society of Chemistry 2016