Cytotoxicity Of Multi-walled Carbon Nanotubes Research Articles

Characterization of short-length multi-walled carbon nanotubes and cytotoxicity on MDA-MB-231 and HUVEC cell lines

Multi-walled carbon nanotubes (MWNTs) are suitable for delivering large biomolecules with lower cytotoxicity values and low prime cost. Surface modifications of MWNTs affect interaction with cells and proteins. Oxidation with strong acids decreases cytotoxicity of CNTs and increases protein-loading capacity. Here, after oxidation, TEM images revealed more aligned structure and carboxylated groups at the surface which decreases toxicity. Functionalized MWNTs showed more gradual degradation than the pristine MWNTs and mass loss increased by 2% in the same temperature range. Raman spectroscopy corrected graphitic structure with characteristic D and G bands at 1330 and 1579 cm−1 and increased intensity after oxidation. FTIR spectroscopy peaks at 1443 cm−1, 1560, 1640 cm−1, 2100–2200 cm−1 and 3426 cm−1 are ascribed to C–O–C vibrational stretch, C=C bonds, vibration of C≡C bonds and stretch of hydroxyl groups, respectively. The sonication-driven dispersion of in phosphate-buffered saline, distilled water and cell culture medium were detected by UV–vis–NIR spectroscopy, water-dispersed functionalized MWNTs revealed the highest absorbance value. Cytotoxicity of MWNTs was investigated before and after functionalization in breast cancer (MDA-MB-231) and human vein endothelial (HUVEC) cells. Relatively low-toxicity results were obtained in functionalized MWNTs and cellular uptake of MWNTs were corrected with fluorescent imaging of cells and cell lysates. Protein-loading capacity of fsMWNTs (functionalized short-length multi-walled carbon nanotubes) was evaluated by using bovine serum albumin (BSA) and with an equal amount of fsMWNTs and BSA; 36% binding yield was obtained. Protein corona after covalent functionalization potentially lowered cytotoxicity up to 6%.

Effect of iron overload from multi walled carbon nanotubes on neutrophil-like differentiated HL-60 cells

Multi walled carbon nanotubes (MWCNTs) are one of the most intensively explored nanomaterials because of their unique physical and chemical properties. Due to the widespread use of MWCNTs, it is important to investigate their effects on human health. The precise mechanism of MWCNT toxicity has not been fully elucidated. The present study was designed to examine the mechanisms of MWCNT toxicity toward human promyelocytic leukemia HL-60 cells. First, we found that MWCNTs decreased the viability of neutrophil-like differentiated HL-60 cells but not undifferentiated HL-60 cells. Because neutrophil-like differentiated HL-60 cells exhibit enhanced phagocytic activity, the cytotoxicity of MWCNTs is dependent on the intracellularly localized MWCNTs. Next, we revealed that the cytotoxicity of MWCNTs is correlated with the intracellular accumulation of iron that is released from the engulfed MWCNTs in an acidic lysosomal environment. The intracellular accumulation of iron was repressed by treatment with cytochalasin D, a phagocytosis inhibitor. In addition, our results indicated that iron overload enhanced the release of interleukin-8 (IL-8), a chemokine that activates neutrophils, and subsequently elevated intracellular calcium concentration ([Ca2+]i). Finally, we found that the sustained [Ca2+]i elevation resulted in the loss of mitochondrial membrane potential and the increase of caspase-3 activity, thereby inducing apoptotic cell death. These findings suggest that the iron overload caused by engulfed MWCNTs results in the increase of IL-8 production and the elevation of [Ca2+]i, thereby activating the mitochondria-mediated apoptotic pathway.

Quasi-SMILES-Based Nano-Quantitative Structure-Activity Relationship Model to Predict the Cytotoxicity of Multiwalled Carbon Nanotubes to Human Lung Cells.

Quantitative structure-activity relationship (QSAR) models for nanomaterials (nano-QSAR) were developed to predict the cytotoxicity of 20 different types of multiwalled carbon nanotubes (MWCNTs) to human lung cells by using quasi-SMILES. The optimal descriptors, recorded as quasi-SMILES, were encoded to represent the physicochemical properties and experimental conditions for the MWCNTs from 276 data records collected from previously published studies. The quasi-SMILES used to build the optimal descriptors were (i) diameter, (ii) length, (iii) surface area, (iv) in vitro toxicity assay, (v) cell line, (vi) exposure time, and (vii) dose. The model calculations were performed by using the Monte Carlo method and computed with CORAL software ( www.insilico.eu/coral ). The quasi-SMILES-based nano-QSAR model provided satisfactory statistical results ( R2 for internal validation data sets: 0.60-0.80; R2pred for external validation data sets: 0.81-0.88). The model showed potential for use in the estimation of human lung cell viability after exposure to MWCNTs with the following properties: diameter, 12-74 nm; length, 0.19-20.25 μm; surface area, 11.3-380.0 m2/g; and dose, 0-200 ppm.

Preparation, characterization and toxicity evaluation of [formula omitted] and NiO-filled multi-walled carbon nanotubes loaded to chitosan

Multi-walled Carbon nanotubes (MWCNTs) have been extensively explored for a variety of biomedical and tissue engineering applications. Their high surface area provides tunable multiple attachment sites for acquiring appropriate biological and biomechanical properties. Despite, the cytotoxicity of MWCNTs is dubious with or without surface functionalization. In the present study, two different kinds of multi-walled carbon nanotubes (MWCNTs) in the presence of cobalt oxide (Co3O4) and nickel oxide (NiO) nanoparticles have been prepared through a simplistic and effective method, and thus, the Co3O4/MWNT and NiO/MWNT structures are loaded to chitosan in methanol solution at 65°C. Results upon the synthesis and structural characterization were obtained by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM), respectively. It has been shown that the tuned functionalized MWCNTs are able to reduce cytotoxic effects and improve biocompatibility and be more appropriate for potential future clinical applications.

Evaluation of Complex Toxicity of Canbon Nanotubes and Sodium Pentachlorophenol Based on Earthworm Coelomocytes Test

As a standard testing organism in soil ecosystems, the earthworm Eisenia fetida has been used widely in toxicity studies. However, tests at the individual level are time- and animal-consuming, with limited sensitivity. Earthworm coelomocytes are important for the assimilation and elimination of exogenous compounds and play a key role in the processes of phagocytosis and inflammation. In this study, we explored an optimal condition to culture coelomocytes of E. fetida in vitro and investigated the cytotoxicity of multiwalled carbon nanotubes (MWCNTs) and sodium pentachlorophenol (PCP-Na) using coelomocytes via evaluating lethal toxicity, oxidative stress, membrane damage, and DNA damage. The results showed that coelomocytes can be successfully cultured in vitro in primary under the RPMI-1640 medium with 2–4×104 cells/well (1–2×105 cells/mL) in 96-well plates at 25°C without CO2. Both MWCNTs and PCP-Na could cause oxidative damage and produce ROS, an evidence for lipid peroxidation with MDA generation and SOD and CAT activity inhibition at high stress. The two chemicals could separately damage the cell membrane structure, increasing permeability and inhibiting mitochondrial membrane potential (MMP). In addition, our results indicate that PCP-Na may be adsorbed onto MWCNTs and its toxicity on earthworm was accordingly alleviated, while a synergetic effect was revealed when PCP-Na and MWCNTs were added separately. In summary, coelomocyte toxicity in in vitro analysis is a sensitive method for detecting the adverse effects of carbon nanotubes combined with various pollutants.

Antibacterial activity and cytotoxicity of multi-walled carbon nanotubes decorated with silver nanoparticles

Recently, various nanoscale materials, including silver (Ag) nanoparticles, have been actively studied for their capacity to effectively prevent bacterial growth. A critical challenge is to enhance the antibacterial properties of nanomaterials while maintaining their biocompatibility. The conjugation of multiple nanomaterials with different dimensions, such as spherical nanoparticles and high-aspect-ratio nanotubes, may increase the target-specific antibacterial capacity of the consequent nanostructure while retaining an optimal biocompatibility. In this study, multi-walled carbon nanotubes (MWCNTs) were treated with a mixture of acids and decorated with Ag nanoparticles via a chemical reduction of Ag cations by ethanol solution. The synthesized Ag-MWCNT complexes were characterized by transmission electron microscopy, X-ray diffractometry, and energy-dispersive X-ray spectroscopy. The antibacterial function of Ag-MWCNTs was evaluated against Methylobacterium spp. and Sphingomonas spp. In addition, the biocompatibility of Ag-MWCNTs was evaluated using both mouse liver hepatocytes (AML 12) and human peripheral blood mononuclear cells. Finally, we determined the minimum amount of Ag-MWCNTs required for a biocompatible yet effective antibacterial treatment modality. We report that 30 μg/mL of Ag-MWCNTs confers antibacterial functionality while maintaining minimal cytotoxicity toward both human and animal cells. The results reported herein would be beneficial for researchers interested in the efficient preparation of hybrid nanostructures and in determining the minimum amount of Ag-MWCNTs necessary to effectively hinder the growth of bacteria.

Culture medium type affects endocytosis of multi-walled carbon nanotubes in BEAS-2B cells and subsequent biological response

We examined the cytotoxicity of multi-walled carbon nanotubes (MWCNTs) and the resulting cytokine secretion in BEAS-2B cells or normal human bronchial epithelial cells (HBEpCs) in two types of culture media (Ham’s F12 containing 10% FBS [Ham’s F12] and serum-free growth medium [SFGM]). Cellular uptake of MWCNT was observed by fluorescent microscopy and analyzed using flow cytometry. Moreover, we evaluated whether MWCNT uptake was suppressed by 2 types of endocytosis inhibitors. We found that BEAS-2B cells cultured in Ham’s F12 and HBEpCs cultured in SFGM showed similar biological responses, but BEAS-2B cells cultured in SFGM did not internalize MWCNTs, and the 50% inhibitory concentration value, i.e., the cytotoxicity, was increased by more than 10-fold. MWCNT uptake was suppressed by a clathrin-mediated endocytosis inhibitor and a caveolae-mediated endocytosis inhibitor in BEAS-2B cells cultured in Ham’s F12 and HBEpCs cultured in SFGM. In conclusion, we suggest that BEAS-2B cells cultured in a medium containing serum should be used for the safety evaluation of nanomaterials as a model of normal human bronchial epithelial cells. However, the culture medium composition may affect the proteins that are expressed on the cytoplasmic membrane, which may influence the biological response to MWCNTs.

Different cellular response mechanisms contribute to the length-dependent cytotoxicity of multi-walled carbon nanotubes

To date, there has not been an agreement on the best methods for the characterisation of multi-walled carbon nanotube (MWCNT) toxicity. The length of MWCNTs has been identified as a factor in in vitro and in vivo studies, in addition to their purity and biocompatible coating. Another unresolved issue relates to the variable toxicity of MWCNTs on different cell types. The present study addressed the effects of MWCNTs' length on mammalian immune and epithelial cancer cells RAW264.7 and MCF-7, respectively. Our data confirm that MWCNTs induce cytotoxicity in a length- and cell type-dependent manner. Whereas, longer (3 to 14 μm) MWCNTs exert high toxicity, especially to RAW264.7 cells, shorter (1.5 μm) MWCNTs are significantly less cytotoxic. These findings confirm that the degree of biocompatibility of MWCNTs is closely related to their length and that immune cells appear to be more susceptible to damage by MWCNTs. Our study also indicates that MWCNT nanotoxicity should be analysed for various components of cellular response, and cytotoxicity data should be validated by the use of more than one assay system. Results from chromogenic-based assays should be confirmed by trypan blue exclusion.

A comparative study of cellular uptake and cytotoxicity of multi-walled carbon nanotubes, graphene oxide, and nanodiamond

AbstractInvestigations of the interactions between carbon nanomaterials (CNMs) and living organisms and their subsequent biological responses are of fundamental significance for toxicity assessment and further biomedical applications. In this work, the cellular uptake and cytotoxicity of multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO) and nanodiamond (ND) were examined and compared. We demonstrated that all of the CNMs were readily internalized by HeLa cells through nonspecific cellular uptake. Their cell uptake ratios showed significant differences in the following order: ND > MWCNTs > GO. A series of biological assays were used to evaluate the cytotoxicity of CNMs. It was found that CNMs showed dose- and time-dependent cytotoxicity to HeLa cells. However, cytotoxicity of CNMs was not associated with their cell uptake ratios. Among them, ND exhibited the highest cell uptake ratio and the least cytotoxicity. To the best of our knowledge, this is the first study which has quantitatively determined and compared the cell uptake ratios and cytotoxicities of MWCNTs, GO and ND. And we expect that these results described here could provide useful information for the development of new strategies to design efficient drug delivery nanocarriers and therapeutic systems as well as deep insights into the mechanism of CNMs' cytotoxicity.

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC 50 of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17–18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.

Cytotoxicity of multi-walled carbon nanotubes in three skin cellular models: Effects of sonication, dispersive agents and corneous layer of reconstructed epidermis

The effects of multi-walled carbon nanotubes were investigated in SZ95 sebocytes, IHK keratinocytes and reconstructed human epidermises. Carbon nanotubes were subjected to dispersion protocols leading to different agglomeration states. Toxicological methods were chosen and adapted in order to ensure compatibility with nanotubes. Results show that: (i) Water-suspended nanotubes, as micrometric agglomerates, were not harmful to skin cells, except minor effects in keratinocytes, (ii) mild sonication slightly decreased nanotube agglomeration but increased cytotoxicity on keratinocytes, (iii) addition of hydroxypropylcellulose or Pluronic F108, which improved nanotube dispersion, masked the harmful effects of sonicated nanotubes. Altogether, these results indicate that carbon nanotubes induced cytotoxicity in human keratinocytes after a short exposure (24–48 h), particularly when they were sonicated before cell incubations. However, the cytotoxic effects of raw and sonicated nanotubes could be prevented in presence of dispersive agents. No cytotoxic effects were observed in SZ95 sebocytes or in stratified epidermises reconstructed in vitro.

Realtime Cytotoxicity Characterization with Cell-Microelectronic Sensing of Water Soluble Carbon Nanotubes

Continuous cytotoxicity characterization of water-soluble carbon nanotubes (CNTs) was reported using a real time celluar analyzing (RTCA) system. The method, which involved the use of microelectrodes-embedded E-plate microwells seeded with cells, was successfully applied to dynamically monitor three water soluble CNTs cytotoxicity towards mouse 3T3 cells. The results demonstrated that cytotoxicity was caused by exposure to individual CNTs, but bundles and single-walled carbon nanotubes (SWNTs) showed more cytotoxicity with much lower IC50 values than multi-walled carbon nanotubes (MWNTs). Moreover, the cytotoxicity of MWNTs was decreased when MWNTs were modified with L-Lysine (MWNT-Lys). Conventional MTT assay validated the results.

The degree of functionalization affects in vitro cytotoxicity of multi-walled carbon nanotubes (CNTs)

The degree of functionalization affects in vitro cytotoxicity of multi-walled carbon nanotubes (CNTs)

Influence of purity and surface oxidation on cytotoxicity of multiwalled carbon nanotubes with human neuroblastoma cells

There are conflicting data concerning the safety and biocompatibility of carbon nanotubes (CNTs). In some reports CNTs have been used for gene delivery without significant toxicity, whereas in others various cytotoxic effects were observed, including induction of intracellular reactive oxygen species (ROS), DNA damage, and apoptosis. Although it is clear that CNT production methods, purity, and functionalization treatments impact on biocompatibility, most of the published reports lack detailed characterization of the CNT samples used. We investigated the effect of various physicochemical features of multiwalled carbon nanotubes (MWCNTs) on toxicity and biocompatibility with cultured human neuroblastoma cells by using MTT, WST-1, Hoechst, and oxidative stress assays. In vitro experiments confirm that after 3 days of incubation with three different types of CNTs dispersed in Pluronic F127 solution, 0.01% cell viability is not affected and apoptosis and ROS are not induced in the SH-SY5Y cells. With prolonged cultures and continued propagation in the presence of MWCNTs, the loss of cell viability was minimal for pure MWCNTs (99% purity), but cell proliferation decreased significantly for 97% purity MWCNTs and acid-treated MWCNTs (97% purity, surface oxidation 8%); no intracellular ROS were detected. When the concentration of CNTs increases, purity and surface oxidation seem to affect cell viability (ED 25 is 48, 34.4, and 18.4 μg/mL, respectively, for 99% purity MWCNTs, 97% purity MWCNTs, and acid-treated 97% purity MWCNTs. Our results indicate that concentrations of 5–10 μg/mL MWCNTs seem ideal for studies on the design and development of artificial MWCNT nanovectors for gene and drug therapy against cancer. From the Clinical Editor With prolonged cultures, loss of cell viability was minimal for preparations with 99% purity, but significant adverse effects were detected with 97% purity and with acid-treated preparations. A concentrations of 5–10 μg/mL of MWCNTs seems ideal for gene and drug therapy against cancer.

Dependence of the cytotoxicity of multi-walled carbon nanotubes on the culturemedium

This study examined the influence of multi-walled carbon nanotubes (MWNTs) on thegrowth of the unicellular protozoan Tetrahymena pyriformis. Contrary to thefindings from most other investigations, our experiment indicated that MWNTsstimulated growth of the cells cultured in proteose peptone yeast extract medium(PPY). Atomic force microscopy images and thermogravimetric analysis showed thespontaneous formation of peptone–MWNT conjugates in the medium by noncovalentbinding. Uptake of large amounts of the conjugates by Tetrahymena pyriformis wasresponsible for growth stimulation, evidenced by images with fluorescently labelledpeptone. After the PPY medium was replaced by a filtrated pond water medium(FPW), however, inhibition of the growth of cells exposed to MWNTs occurred.Measurements of the level of malondialdehyde and superoxide dismutase activitydemonstrated further that MWNTs might be either toxic or nontoxic, depending on themedium used to cultivate Tetrahymena pyriformis. The biological effects of theinteraction of MWNTs with some composites in culture media would be helpfulfor understanding the mechanisms of the toxicity of carbon nanotubes to livingsystems.

Influence of length on cytotoxicity of multi-walled carbon nanotubes against human acute monocytic leukemia cell line THP-1 in vitro and subcutaneous tissue of rats in vivo

Carbon nanotubes (CNTs) are single- or multi-cylindrical graphene structures that possess diameters of a few nanometers, while the length can be up to a few micrometers. These could have unusual toxicological properties, in that they share intermediate morphological characteristics of both fibers and nanoparticles. To date, no detailed study has been carried out to determine the effect of length on CNT cytotoxicity. In this paper, we investigated the activation of the human acute monocytic leukemia cell line THP-1 in vitro and the response in subcutaneous tissue in vivo to CNTs of different lengths. We used 220 nm and 825 nm-long CNT samples for testing, referred to as "220-CNTs" and "825-CNTs", respectively. 220-CNTs and 825-CNTs induced human monocytes in vitro, although the activity was significantly lower than that of microbial lipopeptide and lipopolysaccharide, and no activity appeared following variation in the length of CNTs. On the other hand, the degree of inflammatory response in subcutaneous tissue in rats around the 220-CNTs was slight in comparison with that around the 825-CNTs. These results indicated that the degree of inflammation around 825-CNTs was stronger than that around 220-CNTs since macrophages could envelop 220-CNTs more readily than 825-CNTs. However, no severe inflammatory response such as necrosis, degeneration or neutrophil infiltration in vivo was observed around both CNTs examined throughout the experimental period.