*3.3. Chemical Surface Properties*

The XPS element and atomic concentration survey (Figure 4a) showed that the presence of Si2p is the highest from the blank sample due to utilization of a quartz filter. The concentration fraction of O1s slightly went up in 600 ◦C samples, and significantly increased in the 800 ◦C sample compared to others, indicating that the oxygen increased. The XPS carbon (C1s) spectra of the five samples are depicted in Figure 4b, and are de-convoluted into four peaks. The C–C bond, or graphitic carbon, was dominant, represented by the green peak at approximately 284.2 eV [31–33]. Other peaks at higher binding energies were related to carbon-oxygenated functional groups, including: C–O at 285.6 eV (red peak), –C–OH at 286.8 eV (pink peak), and –C=O at 288.7 eV (blue peak) [30–32]. The component fractions were assessed as functions of furnace temperature in Figure 4c. The C–C content decreased from 66% to 59%, while C–O increased from 16.4% to 19%, and C=O increased from 5.5% to 9.5% with increasing treatment temperature from RT to 800 ◦C. This change indicated that the percentage of oxygen functional groups had increased, resulting from higher amounts of oxygen present on the sample surfaces at higher temperatures. We assumed that the oxidation of graphitic carbon occurred above 600 ◦C.

**Figure 3.** (**a**) SEM and (**b**) TEM images of aBC generated at various treatment temperatures, and (**c**) average particle size distribution for synthesized aBC.

**Figure 4.** (**a**) Element and atomic concentration survey; (**b**) the C1s bond fraction indicates the variation in C–C, C–O, C–OH, and C=O content with increasing treatment; and (**c**) XPS spectra of synthesized aBC samples treated at five different temperatures.

To verify the oxidation of aBC, vibrational characterization was performed by Raman and FTIR analysis. Two typical overlapping peaks are visible in the Raman spectra displayed in Figure 5a, namely a D peak at 1340 cm<sup>−</sup><sup>1</sup> and a G peak at 1600 cm<sup>−</sup><sup>1</sup> [34–37]. The D band represents in-plane breathing vibrations of the aromatic ring structures (A1g symmetry), and the G band is the in-plane stretching vibration of sp<sup>2</sup> carbons (E2g symmetry) [38,39]. The intensity ratios between the D and G bands (I(D)/I(G)) were 0.80, 0.82, 0.82, 0.84, and 0.96 at RT, 200 ◦C, 400 ◦C, 600 ◦C, and 800 ◦C, respectively. The increase in the I(D)/I(G) ratio at 800 ◦C was ascribed to an increase in the in-plane breathing vibrations of the aromatic ring resulting from the appearance of a functionalized group on the aromatic ring. This result provides conclusive evidence of aBC oxidation by thermal processing, especially at 800 ◦C.

**Figure 5.** (**a**) Raman and (**b**) FTIR spectra obtained for five aBC samples generated at specific treatment temperatures.

Figure 5b shows the FTIR spectra of the five analyzed samples. No significant changes were observed among the spectra of the three samples treated in the range of RT to 400 ◦C. These spectra presented a sharp peak at approximately 1635 cm<sup>−</sup>1, assigned to aromatic C=C. Three vibrations located at 2852, 2922, and 2962 cm<sup>−</sup><sup>1</sup> were assigned to asymmetric and symmetric C-H stretching of CH3 and CH2 aliphatic groups [40,41]. At 600 ◦C, the spectrum included an additional carbonyl C=O stretching (1720 cm<sup>−</sup>1) shoulder, becoming a notable peak at 800 ◦C and indicating the presence of oxygen functionalities [41,42].

### *3.4. In Vitro Toxicity of aBC*

Exposure to aBC results in harmful effects on human health, causing pulmonary and cardiovascular diseases [43,44]. The aBC is internalized by various immune and structural cell types, such as macrophages, lymphocytes, skin keratinocytes, and epithelial cells [45–47]. We deduced that aBC would be predominantly deposited in the alveolar region of the human lung by analyzing the electrical current carried by aBC (Figure 6). The estimation

of the lung deposited surface area distribution based on the current charge carried by aBC implied that a higher fraction (50%) of aBC is possibly deposited in the alveolar region than on other regions of the human respiratory system. Alveolar macrophages and alveolar epithelial cells are highly likely to be the primary and secondary targets for aBC if inhaled by humans.

**Figure 6.** Deposited surface area distributions of aBC in three regions of the human respiratory tract, as predicted based on the electrical current recorded by ELPI+.

Herein, we selected the A549 human lung alveolar basal epithelial cell line for in vitro testing. To verify whether aBC directly affects epithelial cells, we confirmed endocytosis of aBC in A549 cells. Diff-Quik staining indicated the appearance of aBC in the cytoplasm and in the region near the nucleus (Figure 7), indicating the appearance of aBC in the cytomorphologic evaluation; however, to confirm the cellular uptake of aBC, further investigation is needed. The particle size is known to have significant effects on the interactions between nanoparticles and the cellular environment [13]. Here, nano-sized aBC has a greater opportunity for cellular uptake via endocytosis by membrane wrapping due to its effective binding to membrane receptors. When aBC interacts and penetrates through the membrane, the defense mechanism is activated, and cell damage occurs [48,49]. In addition, to determine the cytotoxicity of aBC, we evaluated cell viability using the MTT assay. Cytotoxic effects of the particles were clearly observed in epithelial cells 48 h after stimulation with aBC. The results revealed that aBC exhibited considerable cytotoxicity to A549 cells at a concentration of 2 mg/mL, reducing the survival rate of A549 cells to 75%, 68.2%, 68.5%, and 65% when synthesized at RT, 200 ◦C, 400 ◦C, and 600 ◦C, respectively. In particular, the 800 ◦C sample induced the strongest cytotoxic effect (cell viability was reduced up to 45%) compared to the naïve control (Figure 8a). Hence, aBC had adverse effects on the survival of A549 cells; aBC directly contacts the cell membrane, inducing membrane stress by disrupting and damaging it, resulting in cell death. Even exposed to a high dose of aBC, the decreased cell viability was still low compared to other literature due to the aggregation of aBC in the cell medium [11,50].

**Figure 7.** Diff-Quik staining images of (**a**) the naïve control (NC) and (**b–f**) A549 cells stimulated by aBC synthesized at five treatment temperatures. Arrows indicate the internalization of aBC in the A549 cell.

**Figure 8.** (**a**) Cell viability and (**b**) ROS production in the naïve control (NC) and synthesized aBC-stimulated A549 cells. Data are presented as the mean ± SD (n = 8). \*\*\* *p* < 0.01 and \*\*\*\* *p* < 0.001 compared to the NC.

Exposure to aBC induces cell damage via oxidative stress mediated by ROS [47,51]. Excess ROS levels trigger oxidative stress that disrupts cellular homeostasis and affects the oxidation of biomolecules, including DNA, lipids, and proteins [44,52]. ROS production caused by aBC in A549 cells was evaluated by measuring the fluorescence intensity of DCF-CytDA. The aBC generated at higher temperatures induced significant changes in ROS levels in A549 cells compared to the naïve controls (Figure 8b). As observed for cytotoxicity, the greatest oxidative stress was demonstrated in the 800 ◦C aBC samples for particle-stimulated A549 cells. These results indicated that synthesized aBC directly induced ROS-mediated human alveolar basal epithelial cell damage. The production of high levels of ROS causes significant damage to DNA, thereby affecting cell survival. The correlation of biomass burning aerosol chemical composition with ROS production was used for investigation, however, due to the complex effect of chemical compositions, soot-induced bio-toxicity with ROS production was barely interpreted. It is claimed that no significant correlation can be figured out in the ROS response with measured chemical compositions, and the aging process of biomass burning samples even drove the toxicity test to be more complicated [53]. Therefore, to minimize the factors on real-world soot, with the important roles played in the bio-toxicity results, we highlighted the ROS response with the change of the chemical surface on controlled aBC.

The physical characterization illustrated a uniform size and shape of aBC synthesized under varying treatment temperatures. On the other hand, Raman and FTIR analysis supported by XPS data led to the conclusion that a greater number of oxygenated functional groups were present on the surface of aBC treated at elevated temperatures. Cell viability and ROS level results indicated that cytotoxicity increased with increasing oxygenated functional group content on the surface of aBC. This agrees with the findings of Das et al., who investigated the correlation between cellular toxicity and oxygenated functional group density on graphene oxide (GO) [48]. They proved that the presence of organic functional groups on the surface of GO affected its interaction with mammalian cells at the "nano–bio" interface, and that an increase in oxygen functional groups rendered GO less biologically inert and resulted in elevated cytotoxicity. We can therefore infer from the present results that an increase in oxygen functional groups on the surface of aBC activates the "nano–bio" interface, thereby facilitating cell membrane disruption by aBC and resulting in higher cytotoxicity. Furthermore, oxidized flame soot increases ROS levels, as reported by A. Holder. This suggests that the increased cytotoxicity of oxidized soot is due to its ability to generate oxidants [50]. Consequently, studies centered on the evaluation of surface chemical properties associated with cytotoxicity sugges<sup>t</sup> that oxygenated functional groups present on the surface of aBC and aBC oxidation are directly related to cell death and oxidative stress.
