In vitro toxicity of carbon nanotubes: a systematic review† Margarita R. Chetyrkina, a Fedor S. Fedorov * a and Albert G. Nasibulin * ab Carbon nanotube (CNT) toxicity-related issues provoke many debates in the scientific community. The controversial and disputable data about toxicity doses, proposed hazard effects, and human health concerns significantly restrict CNT applications in biomedical studies, laboratory practices, and industry, creating a barrier for mankind in the way of understanding how exactly the material behaves in contact with living systems. Raising the toxicity question again, many research groups conclude low toxicity of the material and its potential safeness at some doses for contact with biological systems. To get new momentum for researchers working on the intersection of the biological field and nanomaterials, i.e., CNT materials, we systematically reviewed existing studies with in vitro toxicological data to propose exact doses that yield toxic effects, summarize studied cell types for a more thorough comparison, the impact of incubation time, and applied toxicity tests. Using several criteria and different scientific databases, we identified and analyzed nearly 200 original publications forming a “golden core” of the field to propose safe doses of the material based on a statistical analysis of retrieved data. We also differentiated the impact of various forms of CNTs: on a substrate and in the form of dispersion because in both cases, some studies demonstrated good biocompatibility of CNTs. We revealed that CNTs located on a substrate had negligible impact, i.e., 90% of studies report good viability and cell behavior similar to control, therefore CNTs could be considered as a prospective conductive substrate for cell cultivation. In the case of dispersions, our analysis revealed mean values of dose/incubation time to be 4–5 mg mL À1 h À1 , which suggested the material to be a suitable candidate for further studies to get a more in-depth understanding of its properties in biointerfaces and offer CNTs as a promising platform for fundamental studies in targeted drug delivery, chemotherapy, tissue engineering, biosensing fields, etc. We hope that the present systematic review will shed light on the current knowledge about CNT toxicity, indicate “dark” spots and offer possible directions for the subsequent studies based on the demonstrated here tabulated and statistical data of doses, cell models, toxicity tests, viability, etc. Introduction Carbon nanotubes (CNTs) attracted the tremendous interest of the scientic community because of their diverse applications in electronics, photonics, composite materials, and as part of energy sources and storage systems. 1–3 Landmark papers pub- lished by Iijima, 4,5 where the structure of multiwalled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) was visualized, ignited a great scope of R&D activi- ties. A remarkable combination of physical and chemical properties, intensively studied in the next few years, pushed researchers' interest towards integrating CNTs into biosystems. Therefore, CNTs were also proposed for biomedical applications such as tissue engineering and regeneration, target drug delivery, hyperthermia treatment for selective cancer cell killing, gene therapy, bioimaging, biosensing, as electrodes for neural prosthetics, etc. 6–11 Such great attention of researchers was driven by a unique alliance of nanoscale size and excep- tional mechanical, optical and electrical characteristics that make CNTs attractive for a direct contact with living systems. 12,13 In 2000, scientists, for the rst time, successfully combined the new material with the most sensitive living system, neurons, giving momentum to the relatively innovative cross-disciplinary eld – nanotechnology for biomedical tasks. 14 However, in the following years, a large number of publications also demon- strated a negative impact of CNTs on biosystems related to hydrophobicity of CNTs, low synthesis-to-synthesis reproduc- ibility of the material characteristics and their unclear acute toxic and long-term biological impacts. 15–26 On the contrary, some studies demonstrated non-toxic effects or apparent toxicity of CNTs in contact with biological systems. 27–33 Metal catalyst impurities, CNT structure and geometry all stemming a Skolkovo Institute of Science and Technology, Nobel Str. 3, 143026, Moscow, Russia. E-mail: albert.nasibulin@aalto. b Aalto University, FI-00076, 15100, Espoo, Finland † Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d2ra02519a Cite this: RSC Adv. , 2022, 12, 16235 Received 19th April 2022 Accepted 19th May 2022 DOI: 10.1039/d2ra02519a rsc.li/rsc-advances © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv. , 2022, 12, 16235–16256 | 16235 RSC Advances REVIEW Open Access Article. Published on 31 May 2022. Downloaded on 6/6/2022 12:05:21 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue