**1. Introduction**

Black carbon (BC) is an undesired byproduct from the incomplete combustion of fossil fuels and biomass [1–5]. BC, commonly referring to soot and found in fine particulate matter (PM2.5), is the main component of atmospheric carbonaceous aerosols [1–3]. As an efficient light-absorbing carbonaceous material, BC has been mainly implicated as a short-lived climate forcer [4–6]. Besides its impact on climate change and the ecosystem, BC has also been associated with pulmonary, cardiovascular, and premature death [3,6,7].

BC is characterized as having fractal agglomerates, being insoluble in water or common organic solvents, and being refractory and potential toxic [2,8]. The factor of human health was less concerned with than climate change, however, a number of studies focus on health impact assessments, which have usually been conducted by estimating the related adverse health outcomes of the population based on the concentration of BC [3,9]. Due to the lack of laboratory BC samples and the inability to separate BC from atmospheric particulate matter, to date, no specific toxicity experiment induced by BC has been conducted [10].

On the other hand, engineered carbon nanoparticles with corresponding toxicity were widely carried out [11,12]. Carbon nanoparticles have been shown to induce inflam-

**Citation:** Le, Y.T.-H.; Youn, J.-S.; Moon, H.-G.; Chen, X.-Y.; Kim, D.-I.; Cho, H.-W.; Lee, K.-H.; Jeon, K.-J. Relationship between Cytotoxicity and Surface Oxidation of Artificial Black Carbon. *Nanomaterials* **2021**, *11*, 1455. https://doi.org/10.3390/ nano11061455

Academic Editors: Vivian Hsiu-Chuan Liao, Saura Sahu and Eleonore Fröhlich

Received: 20 April 2021 Accepted: 28 May 2021 Published: 31 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

mation, enhance oxidative stress, and transform cell signaling and gene expression in mammalian cells and organs, and toxic effects arising from their well-defined physical features have been described in numerous studies [13–16]. Cheng et al. tested ultrafine carbon nanopowder and pointed that carbon particles disrupted the keratinocyte differentiation and upregulated inflammation; carbon powder was claimed to mimic ambient ultrafine particles, however, the commercial carbon powder properties are far different from real-world atmospheric particles [17]. Engineered carbon black and BC share some similar features (black appearance, aggregate morphology, and elemental component) and even some common biological responses; therefore, some of the toxicology results have misunderstood carbon black interchangeably with BC [10,18]. Hong et al. [18], based on elemental analysis, claimed that engineered carbon black with a high, pure percentage of elemental carbon is different from BC, therefore, an experiment with BC that is regarded to have intensive toxicity is needed.

The diesel soot particulate matter is produced by the National Institute of Standards and Technology (NIST), which can mimic real-world diesel soot, however, the existence of polycyclic aromatic hydrocarbon (PAH) with other chemical compositions renders the identification of specific parameters responsible for inducing toxicity and the mechanisms by which they cause harm to the human body highly challenging [19]. Moreover, soot samples were also collected from several sources with a complicated mixture in which BC was barely separated [20–22]. With a mixture of inorganic compounds, trace metals, and PAH, it is challenging to determine the key factors of soot that play the main roles in inducing toxicity. Therefore, artificial BC (aBC) is essential for a toxicity test satisfying three principal requirements: (1) representing elementary atmospheric BC; (2) maintaining origin physical properties; and (3) having a surface that is chemically controllable.

Herein, we have synthesized aBC using an aerosol generator. We defined aBC in this study as the ultrafine particles generated from one origin source, graphite, to simplify the test of relation between toxicity and the chemical surface of aBC; aBC must possess a controllable chemical surface and common physical properties. Thermal treatment is a straightforward method for manipulating the chemical properties of aBC surfaces, especially to control oxygen functional group content while maintaining its physical characteristics. Because a dominant cytotoxicity mechanism of ultrafine particles is a cell–particle interaction accompanied by the overproduction of ROS [23–25], ROS generation and related cytotoxicity were the focus of this study. We demonstrated that an increase in oxygenated functional groups on the surface of BC triggered increased cytotoxicity and ROS levels in the human lung cancer cell line (A549).
