Assessment of Cytotoxicity and Genotoxicity Induced by Diesel Exhaust Particles (DEPs) on Cell Line A549 and the Potential Role of Amide-Functionalized Carbon Nanotubes as Fuel Additive

Abstract

Epidemiological studies have consistently linked air pollution to severe health risks. One strategy to reduce the impact of combustion products from engines is adding additives to the fuel. Potential benefits have been observed in terms of performance and emissions, as well as in decreasing fuel consumption. However, the associated emission of particulate matter into the environment may have unforeseen health effects. This study examines the effects of diesel exhaust particles (DEPs) from diesel fuel mixed with amide-functionalized carbon nanotubes (CNTF). The aim is to analyze the properties of DEPs and determine their toxic effects on lung cells. The DEPs were characterized using scanning and transmission electron microscopy, while the polycyclic aromatic hydrocarbons (PAHs) were analyzed through gas chromatography. Various assays were conducted to assess cell viability, apoptosis, oxidative stress, and DNA damage. The addition of CNTF to diesel fuel altered the morphology and size of the particles, as well as the quantity and composition of PAHs. At the cellular level, diesel DEPs induce higher levels of reactive oxygen species (ROS) production, DNA damage, apoptosis, and cytotoxicity compared to both CNTF and diesel–CNTF DEPs. These findings suggest that the nano-additives enhance energy efficiency by reducing pollutants without significantly increasing cell toxicity.

1. Introduction

Epidemiological studies have shown that exposure to air pollution is associated with serious health risks. Cardiovascular diseases, respiratory diseases, and various types of cancer are among the health issues empirically linked to air pollution [1]. The 2015 Global Burden of Disease Study revealed that outdoor exposure to fine particles (PM2.5) is the fifth leading risk factor for death worldwide, causing 4.2 million deaths that year. Moreover, the World Health Organization (WHO) reports that 92% of the world’s population breathes air with dangerously high levels of pollutants [2,3]. Prolonged exposure to these fine particles can lead to lung inflammation, reduced lung function, and the exacerbation of pre-existing respiratory and cardiovascular diseases [4,5].

The transportation sector is responsible for a significant amount of particulate matter (PM) emissions, especially from compression-ignition (CI) engines that predominantly use diesel fuel [6,7]. One potential strategy to improve air quality worldwide and reduce PM emissions is incorporating nanomaterials into diesel fuel. Nanomaterials have exceptional properties, such as a large surface area, excellent stability, high catalytic performance, rapid oxidation rate, and high combustion heat [8]. When mixed with biofuels or liquid diesel, these characteristics result in better engine performance, optimized combustion, and reduced PM emissions [9,10]. A recent comprehensive review [11] shows that CuO, Al₂O₃, multi-walled carbon nanotubes (MWCNT), CeO₂, GO, carbon nanotubes (CNT), and TiO₂ are the most utilized nano-additives in diesel–biodiesel fuel blends. These additives have demonstrated significant improvements in combustion processes. It has been reported that adding CeO₂ nanoparticles to diesel fuel, whose use is increasing in the United States and Europe, reduced fuel consumption by 5% to 8% and the release of PM and unburned hydrocarbons by up to 15% [12].

Nevertheless, the adverse health effects of releasing CeO₂ and other metallic nanoparticles into the environment have been thoroughly documented. Both in vivo and in vitro studies have demonstrated that these nanoparticles primarily impact the respiratory, nervous, endocrine, immune, and reproductive systems [13]. Extensive research has been conducted to understand nanoparticle toxicity mechanisms and identify the contributing factors to their adverse effects. These factors include the generation of reactive oxygen species (ROS), mitochondrial impairment, inflammation, apoptosis, DNA damage, alterations in cell cycle regulation, and changes in epigenetic control [14,15]. For example, research indicates that exposure to TiO₂ nanoparticles leads to the activation of microglia, the production of ROS, and the activation of signaling pathways involved in inflammation and cell death [16,17]. CeO₂ nanoparticles are associated with various risks, such as the induction of cytotoxicity, oxidative stress, and pulmonary inflammation [18]. Therefore, their use should be limited and controlled. Evidence also suggests that exposure to aluminum nanoparticles can lead to increased oxidative stress, inflammatory events, and the degradation of the blood–brain barrier [19].

A promising avenue to mitigate health risks is using nano-additives that do not contain metals in their chemical structure, such as carbonaceous nano-additives. These additives offer comparable benefits in fuel efficiency and pollutant reduction to metallic additives [20]. CNTs are among the most commonly used nanomaterials as additives to diesel and biodiesel fuel. CNTs are commonly produced by the chemical vapor deposition method (CVD) [21] and have remarkable mechanical, electrical, optical, and thermal properties. However, CNTs often agglomerate and exhibit poor dispersion in various fluids and matrices. To address this issue, covalent and non-covalent functionalization methods have been employed to attach functional groups to the surface of CNTs. Covalent functionalization involves creating a covalent bond between functional entities and the carbon framework of the nanotubes, while non-covalent functionalization relies on supramolecular complexation utilizing forces such as van der Waals forces, hydrogen bonds, electrostatic forces, and π-stacking interactions [22]. In the case of covalent functionalization with amide groups, these can be conducted using oxidized CNT and amines via the solvent method [23] or using an amine treatment in the gas phase at a vacuum pressure. This approach demonstrates rapid and efficient results [24]. Acevedo et al. assessed the impact of functionalizing pristine carbon nanotubes with amide groups and evaluated their stability in commercial diesel. They found that the functional groups on the surface of the carbon nanotubes allowed for the stable dispersion to last up to 70 days [20]. This nanomaterial was additionally evaluated in a stationary compression engine. The results showed that the use of CNTF as an additive to diesel decreased ignition delay and PM emissions [25].

Table 1. Comparison of the properties of the fuel and engine performance with different CNTF additives.

Functionalized Carbon Nanotubes	Concentration in Diesel Fuel	Effect on Diesel Fuel Properties	Effect on Diesel Engine	Ref.
N-doped MWCNTs	20, 50, 75, and 100 ppm	Kinematic viscosity decreased by 10.6% on average There is a slight decrease in density.	Increased cylinder pressure (except at 25 ppm) and heat release rate (13.87% for 100 ppm); decreased ignition delay and reduced NOx, CO, UHC, PM, and smoke.	[26]
Amide functionalized MWCNTs	50 and 100 mg/L (in a blend with 10% vol. biodiesel)	There is no significant effect on kinematic viscosity and heating value at 100 mg/L. Increase in cetane index by 3.57% at 100 mg/L.	No impact on thermal efficiency. Increase in CO emissions and decrease in NO and PM by up to 29.4% and 39.8%, respectively.	[25]
MWCNT-OH	30, 60, and 90 ppm (in a blend of 90% diesel, 5% vol. biodiesel, and 4% or 8% vol. ethanol)	Increase in viscosity and density.	Increases in torque and power and decreases in specific fuel consumption. Decreases in CO, UHC (unburned hydrocarbons), and increases in NOx emissions.	[27]
MWCNT-COOH	30, 60, and 90 ppm	For all MWCNT-COOH concentrations (with 5% biodiesel), kinematic viscosity and density slightly decreased.	Increases in power by 13.01% and decrease in specific fuel consumption by 9.48%. CO and UHC decreased by up to 10.26% and 16.33%, respectively.	[28]
NH2-MWCNTs	25, 50, 75, and 100 ppm	Kinematic viscosity decreased (an average drop of 8.95%), while density was only slightly reduced.	-There was a 13.76% increase in the HHR (100 ppm). -At 50% load, there was a 5.19% decrease in brake-specific fuel consumption and a 5.47% increase in brake thermal efficiency (100 ppm). -At 75% load, CO emissions have decreased by 18.28% (75 ppm) and NOx by 28.97% (25 ppm); there has also been a 14.12% decrease in soot (75 ppm).	[29]
MWCNTs-COOH	30, 60, and 90 ppm	Kinematic viscosity and density were only slightly reduced.	Average power increased by 13.01%; Average torque increased by 17.61%; CO decreased by 10.26%; and UHC by 16.33%.	[28]

shows the effects of adding functionalized carbon nanotubes on the properties of the fuel and engine performance, compared to the properties without the addition of nanotubes.

Beyond the advantages of engine combustion, surface functionalization, both covalent and non-covalent, has enhanced the biocompatibility of carbon nanotubes [30]. CNTF typically exhibits superior hydrophobicity and dispersion in biological media, lower immunogenicity, a reduced tendency to agglomerate, and greater biological compatibility compared to non-functionalized CNT [31,32]. However, available in vitro studies on the toxicity of CNTF yield controversial results. Several in vitro studies have shown that cytotoxic effects are lower in CNTF than in non-functionalized ones. For instance, CNTF with carboxyl, phosphorylcholine, and polystyrene groups induces lower production of cytokines, lung inflammation, and fibrosis compared to non-functionalized MWCNT [33]. However, it is important to note that some studies indicate that the functional group, such as acid functionalization, can significantly affect cellular toxicity, causing oxidative stress, damage to lysosomes, mitochondrial dysfunction, inflammation, and genotoxicity in terms of DNA damage [34,35,36]. The conflicting results mentioned above show that the toxicity of MWCNT depends on its physical and chemical properties, such as purity, chemical composition, size, surface area, and functionalization type.

Despite the numerous reported advantages of using nano-additives in diesel engine combustion, few studies have explored the direct correlation between the emissions of these particles and their effects on health. Currently, there are no reports on the biological effects of amide-CNTF after the combustion process. Therefore, despite the recognized benefits of adding amide-CNTF to reduce pollutants, as shown in Table 1, and improve the properties of the fuels for long-term dispersion, it is essential to evaluate their potential toxic effects and identify the physicochemical properties responsible for their toxicity. This study aims to fill this knowledge gap by examining the cytotoxic and genotoxic effects of diesel exhaust particles (DEP) resulting from using stable mixtures of amide and CNTF in diesel fuel.

2. Materials and Methods

The pristine carbon nanotubes (CNTs) obtained from SkySpring Nanomateriales, Inc. (ref 0554CA) were oxidized using a 1:1 volume ratio mixture of H₂SO₄ (96%, Merck, Rahway, NJ, USA) and HNO₃ (65%, Merck). Acylation was carried out using thionyl chloride (≥99%, Merck) and N,N-dimethylformamide (99.8%, Fischer Scientific, Hampton, NH, USA), and amide functionalization was conducted using technical-grade oleylamine. The amide-functionalized carbon nanotubes (CNTF) were obtained following the experimental methodology established by [23] and adapted by [20,25].

The CNTFs were introduced into Colombian commercial diesel. Prior to the experiments, the fuels underwent filtration using a 6 µm filter paper, as no fuel filter was installed before the engine inlet. The concentration of CNTF in the mixtures was 100 mg/L. The CNTF, in the form of a dry powder, was dispersed in diesel. Ultrasound probe dispersion (model EP225-DR, Hielscher, Ringwood, NJ, USA) was utilized to disperse the CNTF.

2.1. DEPs Collection

The DEPs collection utilized a stationary compression ignition engine connected to a synchronous generator (Figure 1). The engine tests were carried out under an engine load of 50% (6 kW) at a constant speed of 1800 rpm in a three-cylinder diesel engine (Yanmar, 3TNE84, Yanmar Holdings Co., Ltd., Tokio, Japan). An electrical resistance test rig was employed to simulate this load.

DEPs were collected using a DEKATI cascade impactor sampler (PM10 impactor) attached to the engine’s exhaust gasses. The DEKATI impactor is based on the inertial classification of aerosol particles. The sample collection process involved a heated sampling probe, a particle impactor, an impactor heating zone, a pump for extracting combustion products, and a volumetric flow controller. The sampling line was heated meticulously to a temperature of 180 °C to avoid vapor condensation. The volumetric flow was maintained at a constant rate of 10 L/min. Figure 1 demonstrates the sampling process. Before collecting DEPs, the engine was warmed up for 10 min to ensure that the operating conditions were in a steady state and to avoid high DEP emissions associated with a cold engine start [37,38]. The DEPs were sampled to obtain particles with an aerodynamic size of less than 2.5 μm. This size of particle has been classified as a class I carcinogen by the International Agency for Research on Cancer due to its negative impact on health, attributed to its small size and chemical composition [39,40]. This justification supports the selection of this particle size for the present study.

2.2. Physicochemical Characterization of DEPs

The structure of DEPs collected from the test engines underwent examination through transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to characterize their carbon composition and particle size distribution, following the procedures reported by [41,42]. A small volume (3 µL) of each DEP solution was deposited onto a copper grid coated with a polymer support film (Structure Probe, Inc., West Chester, PA, USA) for TEM analysis. Following solvent evaporation in a vacuum environment, the particles’ dimensions and morphology were evaluated using a JEOL 1200EX transmission electron microscope (JEOL, Toyko, Japan) at an acceleration voltage of 120 kV. Images were recorded using an 11-megapixel SIS Morada CCD camera and processed using AnalySIS software V2. The histograms were generated by measuring primary particle diameters derived from TEM images. These images were subjected to analysis using the public-domain image processing software ImageJ V. 1.54j. Primary particles with clearly defined boundaries within the PM agglomerates or non-agglomerated particles were identified from the TEM micrographs, and their respective diameters were determined. Specifically, 200 particles were utilized for the diesel mode, while 160 primary particles were employed for the diesel–CNTF. The frequency histograms were generated through the measurement of primary particle diameters derived from TEM images. These images were subjected to analysis using the public-domain image processing software ImageJ. Primary particles with clearly defined boundaries within the PM agglomerates or non-agglomerated particles were identified from the TEM micrographs, and their respective diameters were determined. Specifically, 200 particles were utilized for the diesel mode, while 160 primary particles were employed for the diesel–CNTF.

For SEM analysis, a 6 mm diameter segment of the filters containing the DEP samples was affixed to a dedicated holder using Quick Drying Silver Paint conductive adhesive (Agar, London, UK). Subsequently, the samples underwent a thin gold coating (approximately 10 nm) using a vacuum evaporator (JEE-4X, JEOL, Japan) to ensure conductivity and safeguard against heat-induced damage. Coated samples were inspected using an HRSEM-AURIGA scanning electron microscope (Zeiss, Oberkochen, Germany) operating in secondary electron (SE) mode. The microscope’s settings were configured with a high voltage (HV) of 10 kV and a working distance (WD) of 20 mm.

The chemical analysis of DEPs was made to determine the PAH composition of diesel and diesel–CNTF fuels. Filters from diesel and diesel–CNTF and a control filter were prepared for PAH analysis. Each filter received an internal standard (Pentachloronitrobenzene) and 5 mL of dichloromethane, then stood for 24 h after sealing and shaking. Following 10 min of ultrasound treatment, the filters were removed, and dichloromethane was evaporated to 0.1 mL using nitrogen. Acetone (1 mL) was added, and samples were transferred to vials for PAH analysis. A calibration curve was created for 16 PAH compounds: naphthalene (Naf), acenaphthylene (Acyl), acenaphthene (Ac), fluorene (Fl), pentachloronitrobenzene (PCNB), phenanthrene (Fen), anthracene (Ant), fluoranthene (Flu), pyrene (Pir), benz(a)anthracene (BaA), chrysene (Ch), benzo(k)fluoranthene (Bkf), benzo(a)pyrene (BaP), benzo(b)fluoranthene (BbF), indeno(1,2,3-cd)pyrene (IP), dibenz(a,h)anthracene (DahA), and benzo(g,h,i)perylene (Bper).

2.3. Preparation of Functionalized Nanoparticle and DEP Samples

To prepare a stock solution of 0.5 mg/mL, 0.5 mg of CNTF was weighed and resuspended in 5 mL of PBS, and 1 µL of DMSO was added as a surfactant to facilitate dispersion. This CNTF suspension was sonicated using a Bransonic CPX-952-338R sonicator (Branson Ultrasonics, Brookfield, CT, USA) for 30 min in three cycles of 10 min each to prevent overheating of the sonication bath. Once the dispersion was obtained, dilutions with concentrations of 2.5, 5.0, and 10 µg/mL were prepared as a treatment for the cell line. This study involved collecting diesel and diesel–CNTF DEPs using Emfab 47 mm filters wrapped in aluminum foil and stored at −20 °C until extraction. Before extraction, the filters were placed in amber bottles, moistened, and sonicated in distilled water for 3 h, with intermittent intervals of 15 min. Following sonication, the filters were air-dried for 3 days in the bottles and then weighed to calculate the concentration of DEPs (mg/mL) based on the difference in weight before and after extraction, determined through 3–5 measurements. The extracted DEPs were then stored at −80 °C for future use.

2.4. Cell Culture Maintenance

Human lung carcinoma cells A549 (ATCC # CCL-185) were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Sigma-Aldrich, St. Louis, MO, USA) with 5% fetal bovine serum (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin. A549 cells were exposed to CNTF and DEPS (diesel and diesel–CNTF) at different concentrations (2.5, 5.0, and 10 μg/mL) for 48 h. Extracts stored at −80 °C were used for exposure. Negative and positive controls were included, with cells subsequently incubated for an additional 24 h at 37 °C with 5% CO₂ after treatments.

2.5. Cellular Uptake of CNTF andDEPs Using Transmission Electron Microscopy

Transmission electron microscopy (TEM) confirmed the cellular uptake of CNTF and DEPs. Cells cultured in T25 flasks were treated with 10 μg/mL of DEPs (diesel or diesel–CNTF) for 48 h. Following trypsinization, the A549 cell suspension was fixed with glutaraldehyde. Sample preparation for TEM included chemical fixation, washing, dehydration, embedding in resin, and polymerization. Ultra-thin sections were prepared using an ultramicrotome and stained with heavy metals for visualization in a Tecnai F20 Super Twin TMP Transmission Electron Microscope from FEI Technologies Inc. (Hillsboro, OR, USA).

2.6. Cell Viability Assay

The MTT assay evaluated the cell viability of A549 cells exposed to CNTF or DEPs (diesel and diesel–CNTF). A549 cells were plated in 96-well plates at a density of 2 × 10⁴ cells/well and incubated for 24 h before exposure to different concentrations of nanoparticles and DEPs (diesel and diesel + CNTF) for 48 h. Following exposure, MTT solution (200 μg/mL) was added, and after a 4 h incubation, formazan crystals were dissolved in 200 μL DMSO. The optical density (OD) was measured at 492 nm using a plate reader (BioTek, Synergy H4, Shoreline, WA, USA) to determine the cell viability percentage compared to control cells.

2.7. Cell Apoptosis Assay

A549 cells were exposed to different concentrations of CNTF and DEPs (2.5, 5.0, and 10 μg/mL) for 48 h in six-well plates. After treatment, the cells were harvested and analyzed for apoptosis using the FITC Annexin V/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) and flow cytometry in a cytometer FACScantoII (BD Biosciences, Franklin Lakes, NJ, USA). The analysis, performed with FlowJo™ v10 software (BD Biosciences), identified three cell populations: early apoptotic cells (Annexin V+/PI−), late apoptotic/necrotic cells (Annexin V+/PI+), and necrotic cells (Annexin V−/PI+). The flow cytometry also excluded small debris and collected ten thousand events from the cell population for analysis.

2.8. ROS Assay

A549 cells were cultured in six-well plates for 24 h, then exposed to CNTF and DEPs (diesel and diesel–CNTF) at specified concentrations (2.5, 5.0, and 10 μg/mL) for 48 h. Following exposure, the cells were harvested and treated with 10 μM DCFH-DA (2′,7′-Dichlorofluorescin Diacetate, Merck KGaA, Darmstadt, Alemania) and incubated at 37 °C for 30 min. The fluorescently labeled cells were analyzed using flow cytometry on a FACSCantoII cytometer (BD Biosciences) to measure the signal in the FITC channel (excitation wavelength = 491 nm, emission wavelength = 525 nm). Data analysis was conducted using the FlowJo™ v10 software (BD Biosciences).

2.9. DNA Damage Assay

The comet assay (pH 13) was utilized to evaluate DNA damage in A549 cells exposed to CNTF and DEPs (diesel and diesel–CNTF) at different concentrations for 48 h. Cells were harvested at a concentration of 1 × 10⁶ cells/mL, embedded on slides (in 0.5% low melting-point agarose on slides coated with 1.5% normal melting-point agarose), lysed at 4 °C for 2 h, and subjected to alkaline electrophoresis followed by neutralization (0.4 mmol/L Tris-HCl, pH 7.5) at 4 °C for 10 min. Ethidium bromide staining and fluorescence microscopy imaging (Olympus BX51) were performed to evaluate DNA damage through the Olive Tail Moment (OTM). Data analysis involved the examination of 100 randomly selected cells per sample using the CometScore image analysis system (TriTek Corp., Williamsburg, OH, USA).

2.10. Statistical Analysis

In analyzing the responses of A549 cells to CNTF and DEP exposure, statistical analysis was conducted using GraphPad Prism 8 software for Windows (GraphPad Software, Boston, MA, USA). The statistical comparisons between the experimental and control groups involved using the homogeneity of variance test and Dunnett’s test for independent samples. Additionally, an ANOVA was applied to compare the responses of the test cells with those of the control cells. Statistical significance levels were defined as *** p < 0.001, ** p < 0.01, and * p < 0.05.

3. Results and Discussion

DEPs were collected from an internal combustion engine using either diesel fuel or diesel–CNTF. In general, the formation of particulate material involves several stages, including pyrolysis, nucleation, surface growth, agglomeration, and oxidation. Pyrolysis reactions occur in an oxygen-free atmosphere and cause the breakdown of fuel molecules into precursor molecules such as acetylene, benzene, or gas-phase PAHs. Subsequently, the nucleation process occurs, where the first soot nuclei are formed by aggregating larger PAH molecules. In the surface growth stage, the mass of the nascent soot particles increases by adding species, leading to an increase in the C/H ratio. The particles undergo agglomeration due to particle–particle collisions, leading to an increase in size and a decrease in number. Finally, the oxygen oxidation stage of PAHs and soot occurs, forming CO and CO₂. This stage competes with its formation during combustion [41,42].

Figure 2A,B show that when diesel is used as fuel, chain-like structures and irregularly shaped particle agglomerates are observed, along with larger agglomerates close to 1 μm in size. Conversely, when the diesel–CNTF mixture is employed as fuel, spherules-like structures are formed (as shown in Figure 3A,B), and agglomerates of particles display fractal-like shapes. Figure 4A shows the presence of individual particles with diameters ranging from 10 to 40 nm when diesel is used as fuel. On the other hand, when the diesel–CNTF mixture is employed as fuel, spherules with diameters between 20 and 150 nm are formed. As a result, using a diesel–CNTF mixture yields larger primary particle sizes than diesel. In both fuel cases, these primary particles serve as the basis for forming particles in the accumulation mode.

In the SEM micrographs (Figure 2C,D), it can be seen that many particles are deposited in the filter structure, with a high degree of overlap. Additionally, larger sizes are observed for the diesel–CNTF case compared to when diesel is used as fuel (Figure 3C,D). This result agrees with the one obtained through the TEM technique, which offers higher resolution. The reason for this is that when primary particles collide, they agglomerate and then form larger particles. If these primary particles have a larger diameter in the nucleation mode, it is expected that the higher diameter observed by SEM would correspond to this phenomenon.

Important factors affecting the formation of particulate matter include the temperature and pressure in the cylinder, as well as the effectiveness of the mixing process between fuel and air [42,43]. On the other hand, in this study, the addition of carbon nanotubes plays a significant role by introducing aromatic-type structures into the fuel. The aromatic structures in the fuel are important for soot emissions and affect the particle size distribution, which is related to variations in the carbon/hydrogen ratio, fuel evaporation, and the formation of intermediate radicals caused by the aromatic structures. These radicals can impact the initial stages of soot formation (e.g., pyrolysis and nucleation) as well as the final oxidation stage [44]. Although the C/H ratio is slightly modified with the concentration of CNTF used in this study (100 mg/L), the addition of these additives could mainly affect the initial stages of soot formation by reducing the ignition delay time [25] and providing precursor molecules for soot formation and surface growth.

Aromatics are important for polycyclic aromatic hydrocarbon (PAH) formation and are a significant soot precursor during diesel fuel combustion due to their high unsaturation. Studies have been conducted both on a laboratory scale using flames or with diesel engines to assess how different fuels affect the production of particulate matter [45,46]. These studies have used mixtures of heptane or diesel with aromatic liquids such as benzene, xylene, or tetralin. They have observed an impact on the morphological characteristics and chemical composition of the resulting particulate material. For instance, it was noted that the particle diameter of the diesel/aromatic soot was larger than when pure diesel was used as fuel, which aligns with the findings of the present study [44]. It is important to note that, unlike those studies, the present study involves the use of a solid nano-additive, significantly increasing the complexity of our research.

The data presented in Figure 5 illustrate the concentrations of PAHs measured when using diesel or diesel–CNTF as fuels. Out of the 16 PAHs examined, 8 depicted in the figure were determined to be above the detection limits of the employed method. The literature has documented the presence of these PAHs in studies analyzing heavy-duty vehicles that utilize diesel [47,48] or in areas characterized by high vehicular volume, such as tunnels [49]. These compounds are primarily attributed to incomplete combustion processes. When comparing the use of diesel fuel with and without functionalized carbon nanotubes (CNTF), it was observed that the general concentration of polycyclic aromatic hydrocarbons (PAHs) was higher in the case of diesel alone. Specifically, the relative concentration of PAHs with five aromatic rings (BkF, BbF, BaP, and DahA) accounted for 86.5% of the total PAHs in diesel mode, followed by PAHs with six aromatic rings (BPer) at 7.9%, and the remaining percentage was attributed to PAHs with four rings (Flu, BaA, and Ch). In contrast, in the diesel–CNTF case, PAHs with five aromatic rings still constituted the largest fraction, but at a notably lower value of 66.4% compared to diesel. The semi-volatile fraction of PAHs with four rings accounted for 31.8%. This indicates that adding functionalized carbon nanotubes to the fuel affects the amount and composition of the PAHs, particularly by favoring the semi-volatile fraction.

The combined findings, along with those obtained through SEM and TEM, could indicate that the introduction of CNTF to diesel may facilitate the surface growth of particles by incorporating PAH species. This process results in increased carbon content and particle size. Furthermore, this addition may also decrease the available quantity of PAHs. However, further investigation is warranted due to the intricate mechanisms underlying particle formation from PAHs and the pyrolysis and oxidation reactions inherent in diesel combustion when CNTF is introduced.

Regarding toxicity, Flu and BPer are categorized by the International Agency for Research on Cancer (IARC) as belonging to group 3, which means they are not classifiable as carcinogenic for humans. Conversely, BaA, Ch, BkF, and BbF fall into Group 2B, signifying that they are possibly carcinogenic to humans. DahA is categorized under Group 2A, indicating that it is probably carcinogenic to humans, while BaP is classified in Group 1 as being carcinogenic to humans [50]. In considering the differential toxicity of PAHs, the potency equivalence factor (PEF) was utilized to assess the overall health impact of PAHs in both diesel and diesel–CNTF modes. The methodology outlined by [51] was adopted to determine the equivalent BaP (benzo[a]pyrene) toxicity of the identified PAHs. This analysis revealed a BaPeq (benzo(a)pyrene equivalent) value of 0.065 mg/L for standard diesel and 0.033 mg/L for diesel–CNTF, indicating that the incorporation of CNTF in diesel yields significant health benefits by reducing toxicity, as measured by BaPeq.

3.1. A549 Cellular Uptake of CNTF and DEPs

TEM enabled the ultrastructural study of A549 cells exposed to CNTF and DEPs (diesel and diesel–CNTF) to determine their cellular entry. It was demonstrated that both CNTF and DEPs enter the cells. In Figure 6A, the control cells showed no structural changes, with no vesicle formation, and organelles such as mitochondria maintained their normal shape. On the other hand, CNTF and both types of DEPs were found within phagosomes in the cytoplasmic region (Figure 6B). There were no free DEP or CNTF agglomerates observed in the cytoplasm or nucleus. While no signs of cell death, such as apoptosis, were observed at the ultrastructural level, treatment with diesel DEPs resulted in increased lysosomes and changes in mitochondrial morphology (Figure 6D). Treatment with diesel–CNTF DEPs led to fewer phagosomes but a higher number of lysosomes compared to treatment with diesel DEPs (Figure 6D).

This study demonstrated that CNTF can enter cells, but this alone does not result in significant cytotoxic effects. It was observed that treating cells with CNTF induces structural changes, notably the formation of phagosomes. This finding aligns with other research showing that endocytosis encapsulates CNT within membrane invaginations, which then bud and fold to form endocytic vesicles. These vesicles are transported to specialized intracellular sorting and trafficking compartments [52]. The cellular uptake mechanism of CNT may vary based on its functionalization and size. However, the specific pathways of entry, subsequent trafficking, and intracellular distribution of CNT remain unclear. Ghosh et al. state that carbon nanotubes trigger a stress response upon entering cells, activating autophagy and/or apoptosis-related pathways. The study suggests that while various mechanisms may contribute to CNT-induced pathogenesis, a comprehensive understanding of the relationship between different types of CNT exposures (multi-walled CNT vs. single-walled CNT) and the processes of autophagy, apoptosis, and necrosis in human lung epithelial cells is still lacking [53].

Our results suggest that the entry of CNTF may occur via autophagy, potentially leading to its elimination through the lysosomal pathway. Autophagy is a highly conserved lysosomal degradation pathway that operates at basal levels in all cells [54,55]. Upon internalization, CNT has been detected in lysosomes, cytoplasmic vacuoles, and phagosomes, free in the cytoplasm, near the nucleus, and occasionally piercing the nuclear membrane [56,57,58,59]. However, further studies are needed to evaluate these pathways and better understand the mechanisms involved.

Unlike CNTF, we observed that exposure of A549 cells to DEPs (diesel and diesel–CNTF) significantly impacts their structure and potential functioning, possibly related to mitochondria and lysosomes. Pierdominici et al. characterized the cytotoxicity of DEPs by examining changes in mitochondrial structure and function. They found that, in addition to morphological alterations, DEPs caused a significant loss of membrane potential (∆Ψm) detectable after just 24 h of treatment. However, this loss of ∆Ψm did not correlate with an increase in the percentage of apoptotic or necrotic cells, which remained unchanged in treated cells compared to untreated cells [60]. Like mitochondria, lysosomes are crucial for assessing the effects of DEPs because they process and digest materials through endocytosis of small molecules and cell surface proteins, phagocytosis of large particles such as apoptotic cell remnants and pathogenic bacteria, or autophagy of cytoplasmic contents, including damaged mitochondria, ER, and lysosomes [61,62,63,64]. In a review by Nie et al., it is mentioned that lysosomes play a role in cellular responses to airborne particulate matter (APM) and DEP-induced stimuli, which can lead to the production of inflammatory factors, interfere with autophagy, alter iron homeostasis, and affect various other mechanisms. Consequently, lysosomal dysfunction or increased lysosomal activity may occur [65].

We focused on the role of diesel–CNTF DEP particles to determine their effect, as this has not yet been reported in the existing literature due to their unique physicochemical characteristics. It should be noted that although adding CNTF to diesel alters the physicochemical properties of the crude soot particles formed by combustion [25], both diesel and diesel–CNTF DEPs enter the cells and potentially cause damage. However, an increase in lysosomes in cells treated with diesel–CNTF DEPs might indicate that this elimination mechanism is activated, thereby reducing potential cytotoxic effects. It is advisable to evaluate autophagosomal markers to understand the mechanism and how this pathway is affected by diesel–CNTF DEPs. Additionally, assessing mitochondrial involvement and lysosomal function is recommended.

3.2. Effect of CNTF and DEPs on Cell Viability

Cell viability percentages (%) were measured using the MTT assay after cells were treated with CNTF, DEPs, and diesel–CNTF DEPs and incubated for 48 h with the corresponding treatment at doses of 0, 2.5, 5, and 10 µg/mL. Each concentration was analyzed in two replicates with three repetitions. Figure 7A illustrates the independent reduction in cell viability induced by each treatment. The black bars indicate that CNTF at concentrations of 5 and 10 µg/mL decrease cell viability by 17.2% and 12.8%, respectively, with statistical significance (p < 0.0001 and p = 0.0001) concerning control cells. Notably, the observed behavior is not dose-dependent, as the 5 µg/mL concentration demonstrates the highest induction of cell death. In the orange bars, it is evident that as the concentration of diesel DEPs increases, cell viability decreases, with effects of 13.8%, 19.6%, and 26.4%, respectively, all significant at p < 0.0001. This damage exhibits dose-dependent behavior. Moving to the blue bars, treatments with diesel–CNTF reduce viability by 15.2% and 18.8% for concentrations of 5 and 10 µg/mL, respectively, with a p-value < 0.0001. Here, it is determined that the behavior may be dose-dependent, particularly at higher concentrations. Additionally, adding CNTF to diesel appears to marginally reverse the harmful effects of the emissions (diesel decreases viability by 26.4%, and diesel–CNTF decreases viability by 18.8%).

A comparison between the CNTF treatments and the DEPs, considering the concentrations used (Figure 7B), reveals significant differences. At concentrations of 2.5 and 5.0 µg/mL, diesel demonstrates a more significant decrease in viability compared to CNTF and diesel–CNTF, with p-values of 0.0087 and 0.0046, respectively. Similarly, at 10 µg/mL, a similar trend is observed, where diesel vs. CNTF shows a significant decrease (13.6%) in viability (p < 0.0001). In contrast, diesel vs. diesel–CNTF indicates a significance level of p = 0.0162 (7.6% decrease in viability). No differences are observed at the 5 µg/mL concentration, as this is the point where CNTF begins to affect viability.

These assays demonstrated that CNTF significantly decreased cell viability at the highest concentrations compared to the control. Notably, a slight impairment of cell viability was observed at an intermediate concentration of 5.0 µg/mL. Toxicological studies on CNTs have shown their impact on cell viability across various cell lines. For instance, they reduce viability in the human epithelial cell line A549 [66] and induce chromatin condensation and DNA fragmentation in mesenchymal stem cells [67]. Additionally, their fiber-like structure and persistence in lung tissues raise concerns about potential negative health effects similar to those caused by asbestos fibers [68]. However, Kharlamova and Kramberger suggest that these adverse effects can be mitigated through the chemical functionalization of CNTFs [69]. Similarly, Coccini et al. reported that amide-functionalized carbon nanotubes exhibit reduced cytotoxic effects compared to non-functionalized nanotubes [70]. Chowdhry et al. also found that functionalized CNTs exhibited lower cytotoxicity compared to non-functionalized ones in both in vitro analyses with HEK 293 cells and in vivo assays with zebrafish [71].

Concerning diesel DEPs, it has been found that, independent of the treatment concentration, there is a decrease in cell viability. This may be due to different factors that can interact and contribute to cell death through various mechanisms, including apoptosis, necrosis, autophagy, and other forms of cell death, such as physical damage. When this effect is compared to the toxicity of DEPs reported in other in vitro studies, the findings of our study are supported by several of these, in which it is shown that diesel engine particles induce cell death in murine RAW 264.7, macrophages, and human A549 lung cells [72], murine endothelial cells SVEC4-10 [73], mesothelial cell line (MeT-5A) and mesothelioma cell line (CRL-5820) [74], co-culture of A549 cells and THP-1 monocyte cells [75], human iPSC-derived microglia [76], and as found here in A549 cells [67]. In the above studies, this effect of decreased cell viability is due to one of the death mechanisms.

This study found differences in PAH content, particle distribution, and morphology between diesel DEPs and diesel–CNTF DEPs. Particle size distribution is a crucial parameter for assessing the cytotoxic effects of DEPs, as it provides insights into the distribution of particles in nucleation and accumulation modes, as well as their potential mechanisms of cellular entry [43]. Likewise, the morphology and microstructure of DEPs are essential for a comprehensive understanding of the potential mechanisms of cytotoxicity [77]. Diesel particles form branched aggregates at a microscopic scale, each consisting of tens to hundreds of spherical or nearly spherical primary particles [78]. On the other hand, the chemical composition of DEPs is also very important when measuring their cytotoxic effects. Different studies mention that PAHs can damage cells and that their content varies according to the type of fuel used [79,80].

Based on the above findings, it could be hypothesized that the effect on cell viability induced by diesel–CNTF DEPs remains marginal or similar to that of diesel DEPs. This may be due to the lower PAH content and the more compact morphology compared to diesel; these factors do not result in a highly significant change in cytotoxic effects. Studies on metallic nanoparticles have shown that their biological effects are diminished when mixed with diesel. However, this reduction is not so significant that it becomes almost imperceptible or negligible [81,82,83,84]. These results are similar to those presented here.

3.3. Induction of Apoptosis by Carbonaceous CNTF in the A549 Cell Line

To evaluate the effect of CNTFs and DEPs (diesel and diesel–CNTF) on A549 cells, an apoptosis and membrane integrity assay was performed using annexin and propidium iodide (PI) double staining. This assay was measured via flow cytometry. Figure 8A shows that different concentrations (2.5, 5.0, and 10 µg/mL) of CNTF and DEP treatments did not compromise membrane integrity compared to the negative control. However, some statistically significant differences were observed when comparing treatments across different concentrations. At 2.5 µg/mL, diesel DEPs induce greater membrane damage compared to CNTFs, with a p-value of 0.0480. At 5 µg/mL, diesel DEPs caused significantly more damage than CNTFs (p = 0.0011) and diesel–CNTF DEPs (p = 0.0005). Finally, at a concentration of 10 µg/mL, a similar pattern to the 5 µg/mL concentration was observed. Diesel DEPs induced more damage than CNTFs (p = 0.0030) and diesel–CNTF DEPs (p = 0.0170).

Regarding the induction of apoptosis, Figure 8C shows that diesel DEPs significantly induced apoptosis at concentrations of 5 µg/mL (p = 0.0092) and 10 µg/mL (p < 0.0001). CNTF and diesel–CNTF DEPs did not show observable effects. When comparing treatments based on the concentrations used, some differences became evident. At 2.5 µg/mL, treatment with diesel DEPs induced more apoptotic cells than CNTF (p = 0.0003), while there were no differences with diesel–CNTF DEPs. At 5 µg/mL, the induction of apoptosis by diesel DEPs was significantly higher than both CNTF (p < 0.0001) and diesel–CNTF DEPs (p = 0.0021). Additionally, diesel DEPs induced more apoptosis than diesel–CNTF DEPs (p = 0.0233). A similar pattern was observed at the highest concentration (10 µg/mL). Both diesel and diesel–CNTF DEPs induced a higher number of apoptotic cells compared to CNTF, with p-values of 0.0068 and 0.0017, respectively. Moreover, diesel DEPs caused greater apoptosis compared to diesel–CNTF DEPs at this concentration (p = 0.0384).

Overall, concentration-dependent diesel DEPs induce greater cell membrane damage and apoptosis induction than CNTFs and diesel–CNTF DEPs. CNTFs alone do not affect cells in terms of membrane damage and apoptosis induction. While diesel–CNTF DEPs induce damage, the use of CNTFs in combination with diesel appears to reverse or reduce the effects seen when using diesel DEPs alone.

This study evaluated cell membrane damage induced by CNTFs and DEPs by double staining with Annexin V and PI. In general, it was observed that membrane integrity was maintained in each of the treatments. This coincides with that reported by Ursini et al., who mention that exposure to CNTFs does not damage the cell membrane of A549 cells [85]. With respect to DEPs, it has been established that, generally, the effect of DEPs did not induce a compromise of the cell membrane [79], supporting the results obtained in this study. On the other hand, apoptosis induction by CNTF was found to have a dose-dependent behavior but without statistically significant differences. This relative induction of apoptosis by CNTF aligns with the study by Patlolla et al., which demonstrated a dose-dependent increase in the proportion of apoptotic cells after 48 h of exposure in human dermal fibroblasts [86]. However, another study indicates that CNTF exposure increased the proportion of apoptotic cells compared to untreated cells, starting with a concentration-dependent trend of 10 μg/mL [87]. In this context, Song et al. state that the cytotoxic effects largely depend on the type of functionalization applied to CNTs [87].

The results observed with DEPs align with numerous publications indicating that, compared to untreated cells, exposure to diesel particles leads to a statistically significant, concentration-dependent increase in the number of apoptotic cells [63,64,65,67]. DEPs can cause inflammation and produce ROS, both of which are associated with mitochondrial dysfunction. Cattani-Cavalieri et al., in their study performed in BEAS-2B cells, found that DEPs alter mitochondrial morphology and reduce mitochondrial bioenergetics, affecting respiration, ATP production, and reserve capacity, all leading to THE induction of apoptosis [88]. Alternatively, Lawal discusses how the organic components of DEPs are accountable for these effects in cells, emphasizing the involvement of DEP-induced stress-activated protein kinases in prompting apoptosis in macrophages [89].

Considering the separate results of CNTF and diesel DEPs and the lack of information regarding their potential toxicological effects when used together, the results obtained with diesel–CNTF DEPs suggest that CNTF could help reduce damage related to cell death and membrane integrity, as no statistically significant induction of apoptosis was observed. Taylor-Just and colleagues suggest that functionalization alone is not the important driver of toxicity but rather the purification method used prior to functionalization [90]. Furthermore, Gamboa et al. have shown that incorporating amide-functionalized CNT can effectively reduce pollutant emissions, such as diesel particulates (DEPs), and enhance the thermal efficiency of compression ignition engines (CIE) [25]. The non-induction of apoptosis by diesel–CNTF DEPs is significant, as CNTFs enhance engine performance and reduce harmful emissions when used with diesel. This beneficial effect is evident in our results. Modifying the physicochemical properties of CNT can impact their toxicity [91,92].

3.4. CNTF and DEPs Induced Oxidative Stress Changes

We compared the intracellular ROS production dynamics after exposing the A549 cell line to different concentrations of CNTF and DEPs (diesel and diesel–CNTF) for 48 h using flow cytometry with the ROS-sensitive fluorescent indicator DCFDA. Figure 9A shows that exposure to CNTF did not increase the DCF signal at any of the concentrations evaluated compared to the negative control, indicating no increase in ROS production. However, the DEPs (diesel and diesel–CNTF) induced ROS production at each of the different concentrations used as treatments, with a p-value < 0.0001.

Figure 9B presents the results of the comparison between treatments based on the dose used. Like the previous findings, diesel DEPs caused higher ROS production than CNTFs and diesel–CNTF DEPs, with a p-value < 0.0001. Combining CNTF with diesel significantly reduced ROS production; however, ROS production was still higher than the negative controls. A dose-dependent behavior was observed throughout the study.

The generation of ROS and the resulting oxidative stress can trigger various cellular processes, including DNA damage and apoptosis [93,94]. Nel et al. identified oxidative stress as the primary mechanism of toxicity associated with nanoparticles, attributing it to their small size and large surface area, which promotes ROS generation [95]. Our research found that CNTF did not induce intracellular ROS production, which was confirmed using the cell-permeable dye DCFH-DA. Functionalization of MWCNTs has been suggested as a strategy to potentially reduce ROS production [68,96,97]. In our study, no significant changes in intracellular ROS levels were observed after exposure to various concentrations of CNTF with amides, contrary to findings with metal- or acid-functionalized CNT, which have shown ROS-mediated cytotoxic damage [32,98,99]. These results align with Vijayalakshmi et al. (2023), who demonstrated that functionalized MWCNTs reduced cytotoxic effects by lowering impacts on cell viability and oxidative stress [100].

Another possible reason why the CNTF used in our study did not generate ROS could be related to their cellular uptake characteristics. Studies indicate that CNTF internalization varies among cell types; for example, some cells internalize CNTF through phagocytosis, while others, such as fibroblasts, show deficient phagocytosis or lack the machinery for endocytosis. Additionally, CNTF may enter cells via energy-dependent mechanisms, which do not induce ROS production [101,102,103,104]. Furthermore, the physicochemical properties of CNT, including their manufacture, application, shape, and durability, significantly influence their biopersistence and potential for ROS-dependent toxicity or cytotoxicity [105,106].

ROS has a dual role in regulating biological processes: at low concentrations, it acts as a second messenger in signal transduction, while at high concentrations, it leads to biomolecule oxidation [107]. In DEP-treated A549 cells, we observed a considerable increase in ROS production. This is observable in both diesel and diesel–CNTF DEPs. However, the addition of CNTF considerably reduces this ROS concentration. It is widely recognized that excessive intracellular oxidative stress induced by DEPs can harm cells by oxidizing lipids, proteins, and DNA [108,109]. In this study, we found a correlation between ROS production and DNA damage induced by DEPs. This will be discussed in the test results below. This higher induction of ROS could be due to the PAH content and structure of diesel DEPs, which have been associated as the major inducers of damage at the cellular level in multiple studies [110,111,112]. Our results show that diesel DEPs have a higher content of PAHs and that their elongated and less condensed structure may be the damage inducers. It is important to mention that although diesel–CNTF DEPs also induce ROS production, at the tested conditions, we were able to detect intracellular ROS generation with a 20% reduction when compared to diesel DEPs. In this sense, the PAH content and the shape of diesel–CNTF DEPs may be involved. Here, we found that, compared with diesel DEPs, diesel–CNTF DEPs have a lower PAH content and a less condensed form, which may be associated with the mechanisms of cellular entry and possible impairment in function and structure. Zerboni et al. found that combining metal oxide nanoparticles with diesel reduced the adverse effects of diesel exhaust particles (DEPs) produced after combustion. Their study noted that no intracellular ROS generation was detected, and only DEPs induced a slight, but not significant, increase in ROS levels. Furthermore, the autophagic pathway could be activated in the presence of nanoparticles compartmentalizing harmful compounds in lysosomes for degradation [83]. The use of nanoparticles can induce a reduction in the effects, and if we extrapolate it to carbon nanotubes, we could be observing a similar behavior.

To gain a more comprehensive understanding of how ROS production induced by DEPs impacts cells, additional assays could be conducted to assess the signaling pathways involved, in addition to evaluating damage. Mitochondria are recognized as the primary generators of ROS, yet they are also highly susceptible to its effects [107]. The harmful effects induced by NOx should also be evaluated. Although it has been reported that amide-functionalized CNTF, when used in a mixture with diesel, significantly reduces NOx emissions by up to 29.4% [25], NOx can affect cell metabolism like ROS [103].

3.5. DNA Damage on A549 Cells Treated with CNTF and DEPs

Finally, we examined DNA damage in A549 cells treated with CNTF and DEPs (diesel and diesel–CNTF) using a comet assay to assess their genotoxic impacts. Figure 10A–E shows images of the tested cells and the comets formed in some of them after treatment. Figure 10F presents the results of the comparisons made according to the type of treatment. Treatment with CNTFs did not induce DNA damage at any of the concentrations used. Conversely, diesel DEPs and diesel–CNTF DEPs induce DNA damage, which can be visualized in the tails of the comets formed (D-E) with a significance of p < 0.0001. The comet tails are longer in the diesel DEP treatment at each concentration.

Figure 10G shows the results of the comparisons made according to the different concentrations and types of treatment. In general, it is observed that DEPs induce greater DNA damage than diesel–CNTF particles and CNTF alone. At concentrations of 2.5 µg/mL, 5.0 µg/mL, and 10 µg/mL, DEPs caused the most damage, followed by diesel–CNTF particles, compared to CNTF. Additionally, when comparing the genotoxic effects between diesel DEPs and diesel–CNTF DEPs, it was found that the former induced greater DNA damage, with larger comet tails and statistically significant differences between these two treatments (p = 0.0044). These findings suggest that diesel DEPs exhibit the highest DNA-damaging potential. Furthermore, exposure of the A549 cell line to the functionalized nanoparticles did not increase DNA damage. Although diesel–CNTF DEPs also induced DNA damage, the size of the comets formed was much smaller than that produced by diesel DEPs. As with previous assays, using CNTFs appears to mitigate the damage induced by diesel. The observed comet formation behavior is dose-dependent.

Our study revealed no genotoxic effect in cells exposed to CNTF. These results suggest that CNTFs possibly did not enter the nucleus and interact with DNA. Perhaps after a longer exposure time or an increase in concentration, this effect may be more pronounced. Studies investigating the toxicological effects of functionalized CNTs have primarily utilized carboxylic acid and amide-functionalized CNTs [85,86,87,113]. In their study, Jos and colleagues discovered that exposing differentiated and non-differentiated Caco-2 cells to COOH-CNTs at concentrations ranging from 5 to 1000 μg/mL for 24 h resulted in cytotoxic effects, particularly at concentrations exceeding 100 μg/mL [114]. Similarly, Patlolla et al. observed that the maximum increase in tail DNA was observed in COOH-functionalized (400 μg/mL) at 48 h post-treatment compared to the control [86]. Using low concentrations of CNTF could be a viable alternative to avoid significant biological effects. However, it is recommended to conduct measurements over extended exposure periods.

The effect of the type of functionalization on genotoxicity has been widely evaluated. It has been determined that COOH-CNTs have the highest level of toxicity and that NH2-CNTs have a lower cytotoxic and genotoxic effect [87,115,116]. In addition, other studies have reported that the physiological morphology and structure of CNTs and target cells were one of the main factors in inducing toxicity and genotoxicity [87,117]. This could explain what was found in our study, since the amide functionalization of CNTs and the type of cells used could be reducing their entry and subsequent damage. It has been suggested that early DNA damage highlights the carcinogenic potential of CNTs in lung cells; however, repair mechanisms can restore DNA integrity in most cases [118,119,120,121,122]. This is consistent with the lack of an increase in DNA damage frequency observed in this study in cells treated with CNTF, leading to the hypothesis that DNA breakage induced by these functionalized CNTs is poorly related to potential carcinogenesis.

Our results also indicate that DEPs at each of the concentrations used cause genotoxic damage. However, all concentrations of diesel DEPs differed statistically significantly from diesel–CNTF DEPs. Different studies have shown that DEPs can generate genotoxic damage that has been associated with oxidative damage and DNA strand breaks [65,67,97,99,113]. We found that there is a relationship between increased ROS and DNA damage that is not associated with direct damage to cell structure and function. Among the mechanisms that can generate DNA damage induced by increased ROS produced by DEPs are single and double-strand breaks, abasic sites, base and sugar lesions, and numerous oxidative lesions derived from pyrimidine and purine, such as the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine [123,124,125,126,127,128,129].

Exhaust emissions from the two fuel combinations (diesel or diesel–CNTF) showed different toxicological effects. The general mechanisms of DEP-induced toxicity are complex because of the chemical complexity of DEPs, the biological complexity of the cells involved, and the interactions between them [75]. The chemical composition of DEPs, including the concentration of metals and PAHs, has been shown to impact health due to their ability to induce oxidative stress and disrupt normal homeostasis that can lead to toxicological endpoints—cytotoxicity, genotoxicity, and ultimately cell death [130,131,132,133,134]. In addition, the ability of DEPs to absorb and transport compounds to deeper regions of tissues and cells is also associated with their morphological characteristics, with their size and shape being important aspects to take into consideration [135,136,137,138].

The differences in biological effects between diesel and diesel–CNTF DEPs can be attributed to variations in chemical composition and morphology. When CNTFs are used with diesel, both the composition of PAHs and the morphology change, resulting in a lower hydrocarbon content and a more compact and larger particle morphology. Zhang and Balasubramanian note that incorporating metal nanoparticles into fuels can effectively reduce particle mass concentrations, alter chemical composition, and modify particle shape [84]. Similar findings have been reported by other researchers, indicating that the inclusion of carbon nanotubes or metallic particles can significantly alter the physicochemical characteristics of DEPs, or particulate matter [139,140,141]. Given these observations, future research should focus on investigating the toxicological responses to DEP-CNTF at higher concentrations and extended exposure times. Additionally, evaluating signaling pathways related to DNA damage repair, cell cycle regulation, and cell death induction will be crucial to understanding the broader implications of these modifications.

4. Conclusions

This article presents original research findings pertaining to the impact of DEPs derived from an internal combustion engine utilizing either diesel fuel or a combination of diesel and CNTF. Specifically, this study assesses the effects of these DEPs on cytotoxicity and genotoxicity in lung cells while also examining the physicochemical properties of the DEPs and their correlation with cellular damage. Significantly, the research addresses a gap in the existing literature by focusing on the cellular damage caused by DEPs resulting from the presence of carbonaceous additives (CNTF), an area that has received limited attention compared to studies involving additives containing metallic components.

In the physicochemical characterization, it was observed that the addition of CNTF to diesel fuel altered the morphology and size of particles, as well as the quantity and composition of PAHs. This resulted in the formation of spheroid structures and aggregates of fractal-type particles, with an increase in aerodynamic diameters compared to diesel alone. The concentration of PAHs decreased overall, and the fraction of four aromatic rings (Flu, BaA, and Ch) increased by 31.8% in comparison to the total PAHs in diesel, which only accounted for 5.5% of this fraction. In terms of toxicity, this study identified a BaPeq (benzo(a)pyrene equivalent) of 0.033 mg/L, lower than the 0.065 mg/L found in diesel, suggesting that the incorporation of CNTF in diesel can effectively reduce the toxicity associated with these compounds.

Our research found that diesel DEPs induced higher levels of ROS production compared to both CNTF and diesel–CNTF DEPs. The combination of CNTF with diesel significantly reduced ROS production. We also observed that exposure to DEPs caused genotoxic damage, whereas exposure to functionalized nanoparticles did not increase DNA damage. In addition, the use of CNTF appeared to mitigate diesel-induced damage. We can conclude that the functionalization of carbon nanotubes can potentially reduce the toxic effects of diesel exhaust particles and improve their safety for energy applications.

In conclusion, this investigation provides a deeper understanding of the effects of non-metallic nano-additives on cell toxicity and their potential to improve fuel efficiency. This adds a new perspective to research on fuel nanomaterials, suggesting that amide-functionalized nano-additives could offer a safer alternative to traditional metallic additives with a lower risk of adverse health effects. This approach may influence the development and adoption of new, cleaner, and less toxic fuel technologies.