*2.2. Physicochemical Characterization*

An electrical low-pressure impactor (ELPI+TM, DEKATI, Finland) was employed to measure the concentration of generated particles in the range of 6 nm to 10 μm with a time resolution of 1 s. Furthermore, we estimated the surface area concentration of the deposited particles in the human respiratory tract based on ELPI+ stage-specific conversion factors. Teemu et al. estimated the ELPI+ response coefficient (β) by using an equivalent unit density (1 g/cm3) for spherical particles, with a possible error in the mean diameter arising at each stage of each instrument 2% [26]. From the measured electrical current (I) carried by the aBC, the deposited area concentration of aBC (Adep) was estimated for three regions, head airways, tracheobronchial, and alveolar, according to the following equation:

$$\text{Adep} = \emptyset \times \text{I}$$

The deposited area distribution of aBC was estimated to select suitable cell lines for cytotoxicity testing.

In the second experiment, samples were collected on pre-fired quartz fiber filters (six hours, 600 ◦C) with a diameter of 25 mm (FTQ25, Zefon, Ocala, FL, USA) and Teflon filters (R2PJ047, Pall Corporation, Port Washington, NY, USA) with a diameter of 47 mm using filter holders at five different temperatures (RT, 200 ◦C, 400 ◦C, 600 ◦C and 800 ◦C); these samples were utilized for chemical analysis and the in vitro test.

High-resolution transmission electron microscopy (HR-TEM; JEM-2100, JEOL, Akishima, Japan) was employed to observe the size and morphology of the aBC at an accelerating voltage of 200 kV. The aBC was collected on holey carbon TEM grids using a mini particle sampler (MPS, Ineris, Ecomesure, Saclay, France). For SEM analysis, aBC was dispersed on a heated (100 ◦C) silicon wafer by depositing a drop of particle suspension, created by sonicating aBC collected on a quartz filter in DI water. These samples were coated with a thin film of Pt and then observed by field emission-scanning electron microscopy (FE-SEM; S-4300, Hitachi, Tokyo, Japan).

The surface chemical compositions of the aBC samples were analyzed by X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific Co., Walthem, MA, USA). Additionally, Raman spectra were acquired using an Xplora spectrometer (Horiba Jobin-Yvon, Palaiseau, France) equipped with a 532 nm solid state laser source. Fourier transform infrared vacuum spectroscopy (FTIR; Bruker VERTEX 80V, Coventry, UK) was employed to investigate structural changes occurring during the heating process.

### *2.3. Endocytosis, Cytotoxicity Assay, and Evaluation of Reactive Oxygen Species (ROS)* 2.3.1. Cell Culture

A549 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Roswell Park Memorial Institute (RABCI) 1640 (Gibco, Grand Island, NY, USA) containing 5% fetal bovine serum (FBS; Gibco) and 1% streptomycin and penicillin at 37 ◦C in an atmosphere containing 5% CO2.
