Toxicity assessment of nano-sized MAX phases: considerations for safe-by-design approaches

Abstract

MAX phases are versatile materials with unique metallic and ceramic properties, utilized in aerospace, nuclear engineering, and high-temperature applications. A comprehensive assessment of the potential hazards and toxicity mechanisms is essential to ensure the safe utilization of these nanomaterials. In light of this, our study investigates the toxicity of two nano-sized MAX phases, Ti₂AlC and Ti₃AlC₂, to provide fundamental data for implementing the safe-by-design (SbD) approach. Cytotoxicity, genotoxicity, and ecotoxicity screening assays were conducted to identify environmental health and safety issues associated with these materials. The comparison with graphene oxide served as a reference nanomaterial in all toxicity tests. At a concentration of 1 mg L⁻¹, MAX phases showed approximately 20% cytotoxicity and a significant increase in DNA strand break marker and IL-6 level in BEAS-2B cells. However, at the same concentration, no significant toxicity was observed in C. elegans and zebrafish embryos. Overall, MAX phases exhibited non-negligible toxicity, with genotoxicity being the most notable endpoint. This study fills the knowledge gap between the prospective use of MAX phases in the biomedical field and their influence on the environment and human health. These findings underscore the importance of evaluating the potential hazards associated with nano-sized MAX phases and provide valuable insights for the implementation of SbD during their research, development, and design phases.

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Environmental significance

The potential release of nano-sized MAX phases in various applications raises concerns about their environmental exposure and potential hazards. In response, our study investigated the cytotoxicity, genotoxicity, and ecotoxicity of two representative MAX phases, Ti₂AlC and Ti₃AlC₂, shedding light on their potential risks. Our findings reveal significant cytotoxicity and genotoxicity in human bronchial epithelial cells, underscoring the need for cautious consideration of their use. Furthermore, our research contributes vital data for the implementation of the safe-by-design (SbD) approach, providing a proactive strategy for the development of safer nanomaterials.

1. Introduction

A group of ternary carbides and nitrides that exhibit various properties of metallic and ceramic materials, MAX phases have the general formula M_n+1AX_n (n = 1, 2, 3…), where M is an early transition metal, A is a group IIIA or IVA element, and X is either nitrogen or carbon.^1–3 MAX phase materials have various applications, including aerospace, nuclear engineering, and high-temperature applications,^3–5 owing to their appealing material properties, such as good thermal/electrical conductivity, low density, high ductility, toughness, and corrosion resistance.⁶ The MAX phase can also be used as a precursor for synthesizing MXenes, a family of two-dimensional transition metal carbides, nitrides, and carbonitrides. MXenes are materials with potential applications in various fields such as energy storage and conversion, environment and catalysis, separation membranes, medicine, optics, and electronics.⁷ As the application range of MXenes increases, the need for research on the hazards of the MAX phase also increases.

In light of the potential versatility of MAX phases for future applications, extensive research has been undertaken to develop various MAX phase materials. However, the assessment of human and environmental hazards associated with these materials remains understudied.^8,9 However, MAX phases, being nanomaterials, are also presumed to possess potentially harmful properties. For instance, MAX phase and MXene nanomaterials often have similar physicochemical properties and applications comparable to graphene family nanomaterials (GFNs). GFNs, which were developed earlier than MXene,¹⁰ have been extensively studied for their potentially harmful effects in both in vitro and in vivo systems.¹¹ The toxicity of GFNs encompasses physical destruction, oxidative stress, DNA damage, inflammation, apoptosis, autophagy, and necrosis.^11–14 Improvements in the toxicological knowledge of MXenes and their precursor MAX phases, which are relatively meager compared to toxicity studies of GFNs, are needed, which will facilitate their broader application.

The “safe-by-design” (SbD) approach addresses environmental, health, and safety (EHS) issues during the research and development (R&D), and design phases of nanotechnologies.^15,16 To achieve the development of safer MAX phase materials through the application of SbD, an understanding of potential human and environmental impact is essential.¹⁷ In this study, the toxicity of two types of MAX phases, Ti₂AlC and Ti₃AlC₂, were explored to provide a basis for SbD on MAX phases. The status of toxicity studies of MAX phases and MXenes was first confirmed through an analysis of previously published literature. Then, cytotoxicity, genotoxicity, and ecotoxicity screening assays were conducted to identify EHS issues in these materials. Cytotoxicity and genotoxicity assays were performed using human bronchial epithelial cells (BEAS-2B), and toxicity assays using Caenorhabditis elegans and zebrafish were conducted for ecotoxicity screening. All toxicity tests were conducted on Ti₂AlC and Ti₃AlC₂, together with graphene oxide (GO) as a reference nanomaterial.

2. Materials and methods

2.1. Characterization of MAX phases

The commercially available MAX phase particles, Ti₂AlC and Ti₃AlC₂ powders (99.99%), were purchased from Carbon-Ukraine Ltd. (Kyiv, Ukraine). The nano-sized MAX phase particles were obtained by bath-type ultrasonication (Kum Sung, Korea) in 20 mL of distilled water for 16 h. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), energy dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) were performed to determine the material properties of the MAX phase materials (Table 1). FESEM and EDS (SU8010, HITACHI, Japan) were conducted to examine the surface morphology and identify the elemental composition of the MAX phase particles. The particle thickness and lateral size of the MAX phase particles were characterized using AFM (XE-100, Park Systems, Korea). The bonding on the surface of the MAX phase particles was analyzed using XPS (K-alpha plus Thermo Scientific, USA) with an Al Kα line-focused monochromator (1486.6 eV) with a beam size of 200/400 μm. Survey scans were measured at a pass energy of 200 eV and a step size of 1 eV. Structural characterization of the MAX phase particles was performed by XRD (Rigaku, Smartlab, Japan) over the 2θ range of 5–85° at a scan speed of 3° min⁻¹ and a scan step of 0.05°. The ζ-potentials of the MAX phases in the test media were evaluated using a dynamic light scattering (DLS) spectrometer (ELSZ-1000, Otsuka Electronics Co. Ltd., Japan).

Table 1 Physical property analysis information of MAX phases

Methods	Manufacturer (product)	Parameters	Result
FESEM	HITACHI (SU8010)	Surface morphology	Fig. 1(a)
AFM	Park Systems (XE-100)	Particle thickness	Fig. 1(b and c)
EDS	HITACHI (SU8010)	Identification of composition of elements	Fig. 1(d)
XPS	Thermo Fisher Scientific (K-Alpha+)	Analyzing the surface chemistry	Fig. 1(e)
XRD	Rigaku (Smartlab)	Phase identification of a material	Fig. 1(f)
DLS	Otsuka Electronics (ELSZ-1000)	Particle size & stability	Fig. 2

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2.2. Cell culture and treatment of MAX phases

Human bronchial epithelial cells, BEAS-2B, were purchased from the American Type Culture Collection (Manassas, VA, USA). BEAS-2B cells were maintained in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12, GIBCO BRL Life Technologies, Rockville, MD, USA) supplemented with 10% (v/v) fetal bovine serum and 1% antibiotics, at 37 °C in a humidified atmosphere containing air and 5% CO₂. MAX phases (Ti₂AlC and Ti₃AlC₂) were freshly prepared in cell culture medium (DMEM/F12) at the desired concentrations with an appropriate amount from the stock solution (1000 mg L⁻¹ in distilled water). The samples were sonicated for 30 min before biological exposure. In the pristine state, MAX phases are not water-dispersible and thus require treatment with a surfactant, Pluronic-127 (Sigma Aldrich, St. Louis, MO, USA) allow them to disperse in the media. A 0.1% concentration of Pluronic-127 (v/v), which had no adverse effect on the model organisms, was used.

2.3. Cytotoxicity assay

BEAS-2B cells were seeded at a concentration of 1 × 10⁵ cells per mL in 6-well plates and exposed to MAX phases (Ti₂AlC and Ti₃AlC₂) at different concentrations from 0.01 to 1 mg L⁻¹. The treated and untreated cells were incubated for 24 h and harvested for cytotoxicity assays. Cell viability was measured using a RealTime-Glo™ MT cell viability assay (Promega, WI, USA) in a complete DMEM F/12 medium. Three separate experiments were performed for all concentrations in triplicate.

2.4. Comet assay

BEAS-2B cells (5.0 × 10⁴ cells per mL) were seeded in 6-well plates for 24 h before treatment and treated with MAX phases (Ti₂AlC and Ti₃AlC₂) at 1 mg L⁻¹ for the next 24 h. After the treatments, an alkaline (pH > 13) comet assay was performed using the standard method described previously.¹⁸ After exposure, the cells were trypsinized and centrifuged at 1100 rpm (200 × g) for 2 min. Approximately 2–6 × 10⁴ cells were resuspended in 0.5% low-melting-point agarose (Bio-Rad Laboratories, CA, USA) at a 1 : 1 ratio. The resuspended cells were placed on slides pre-coated with 1% normal-melting agarose. After solidification, the slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-base, 1% N-lauroyl sarcosinate, 1% Triton X-100) for 1.5 h at 4 °C. Slides were transferred to an electrophoresis tank containing freshly made electrophoresis buffer (1 mM EDTA, 300 mM NaOH; pH > 13) and kept for 20 min at room temperature to allow DNA unwinding. Electrophoresis was performed in the same buffer at 25 V and 300 mA at room temperature for 20 min. The slides were neutralized three times with 0.4 M Tris-chloride buffer (pH 7.5), air-dried, and fixed in 70% ethanol. The slides were stained with 40 μL ethidium bromide (10 μg mL⁻¹) and analyzed using a fluorescence microscope (Leica DM IL, Switzerland) at 40× magnification. Approximately 50 cells per slide (three slides per treatment) were examined. DNA damage was expressed as the olive tail moment using image analysis Komet 5.5 software (Kinetic Imaging Ltd., UK). Pb²⁺ was used as the positive control.

2.5. Inflammation assay

Interleukin (IL)-6 levels in culture supernatants were measured using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA). The culture supernatants were collected after each experiment, centrifuged at 11 000 rpm for 20 min, and frozen at −80 °C. IL-6 was quantified in each supernatant in duplicate. The optical density was read on a GloMax® microplate reader (Promega, WI, USA) at a wavelength of 450 nm with a reference wavelength of 540 nm. Values were normalized to those of the control group.

2.6. C. elegans toxicity assay

C. elegans was provided by the Caenorhabditis Genetics Center (MN, USA), cultured in Petri dishes on a nematode growth medium at 20 °C, and fed OP50 strain Escherichia coli according to the standard protocol.¹⁹ Wild-type (N2), and nth-1 (ok724) and ndx-4 (ok1003) loss-of-function mutants were used. The toxicity of the MAX phases on C. elegans was investigated by measuring changes in the reproductive potential after chemical treatment. C. elegans reproduction assays were conducted using a Complex Object Parametric Analyzer and Sorter (COPAS™ SELECT, Union Biometrica, Holliston, MA, USA).²⁰ For the reproduction experiments, 24 replicates were used for each treatment.

2.7. Zebrafish toxicity assay

Adult zebrafish, progeny from wild-type fish, were maintained in a flow-through system of carbon-filtered tap water (pH = 7.9, hardness = 6.7 d, conductivity = 468 S cm⁻¹) at 26 °C with a photoperiod of 14:10 h (light:dark). The fish were fed daily with commercial flakes (SERA Vipan, Germany) as a staple feed supplemented with frozen chironomids and frozen Artemia nauplii (Akvarieteknik, Sweden). The fish were placed in cages in aquaria on the afternoon of the day before egg collection. The MAX phase toxicity in zebrafish was investigated by measuring embryo mortality by chemical treatment. The zebrafish embryo test was conducted according to the Organization for Economic Cooperation and Development test guideline no. 236 with slight modifications.²¹ Briefly, newly fertilized zebrafish eggs were exposed to the MAX phases for 96 h. The coagulation of fertilized eggs, a marker of mortality, was evaluated at the end of the exposure period. All procedures were approved by the Institutional Animal Care and Use Committee at University of Seoul, and conducted in accordance with relevant guidelines and regulations in Republic of Korea.

2.8. Statistical analyses

The significance of the differences among the treatments was tested using a one-way analysis of variance followed by a post hoc test (Tukey's test, p < 0.05). All statistical analyses were carried out using IBM SPSS (version 20.0; SPSS Inc., IL, USA), and graphs were prepared in SigmaPlot (version 12.0).

3. Results and discussion

3.1. Application and biocompatibility of MAX phases

To investigate the application and biocompatibility of MAX phases and MXenes, relevant literature was collected from PubMed. The search was conducted using keywords such as ‘MAX phase’, ‘MXene’, ‘toxicity’, and ‘biocompatibility’ (January 2022).

The MAX phase has metallic and ceramic properties; light, stiff, and resistant to fire, oxidation, and radiation.²² Research is underway to utilize the MAX phase with these physicochemical properties in various fields. Boatemaa et al. reported that Ti₃AlC₂ and Ti₂AlC resist oxidation in the 900–1400 °C temperature range,²³ and Wang et al. confirmed the potential of Ti₂AlC to be utilized as a high-temperature solar absorber material.²⁴ The MAX phase has the potential to be applied to nuclear engineering as it is resistant to radiation as well as heat.²⁵ At the evaluation of neutronics and neutron radiation damage response for in-core nuclear applications, Ti₃AlC₂, and Ti₂AlC showed potential,⁴ Whittle et al. also confirmed the radiation tolerance of Ti₃AlC₂.²⁶ Since most of the MAX phases have good electrical conductivity,²⁷ many studies have been conducted to use Ti₃AlC₂, and Ti₂AlC as saturable absorbers.^28–30 Also, Ti₃AlC₂ functions as a catalyst in hydrogen storage properties of MgH₂ or a catalyst for oxidative dehydrogenation of n-butane.^31,32 Overall, given that MAX phases have high application potential in broad fields, the possibility of their exposure to the environment and human body through various media increases, and therefore, a wide range of hazard screening is needed.

In this study, previous studies on the biocompatibility of MAX phases and MXenes were analyzed (Table 2). The number of studies on the biocompatibility of the MAX phase is much smaller (two studies)^33,34 than that of MXene (nine studies).^35–42 Also, no biocompatibility studies on the Ti₃AlC₂ and Ti₂AlC could be found. Most of the studies for the MXene have focused on its applicability for biomedical purposes, and there have been only two toxicity studies. Various methods were used to measure the physical properties of the materials: nine studies using SEM for morphology, six studies using DLS for particle size/stability, and transmission electron microscopy for structure, four studies using EDS for the elemental composition three studies using XPS for surface chemistry, Fourier transformation for layered reflection, and infrared spectroscopy for bond vibrations. In the studies for the MAX phase, the human cervical cancer (HeLa) cell line and human fibroblast (MSU1.1) cell line³³ were used for biomedical application, and rice (Oryza sativa L.) was used for ecotoxicity.³⁴ For the toxicity endpoint, cell viability, reactive oxygen species (ROS), and morphology were measured in the biomedical application study,³³ and growth indicators, antioxidant enzyme expression, and morphology were measured in the ecotoxicity study.³⁴

Table 2 Summary of physical property and toxicity analysis information of MAX phases and MXenes

MAX phases/MXenes	Material characterization		Toxicity		Findings	Ref.
	Methods	Parameters	Model	Endpoint
TiC	SEM	Morphology	Human cervical cancer (HeLa) cell line, human fibroblast (MSU1.1) cell line	Viability	Exposure to high concentrations of TiC and MAX phases <44 μm in size induces cytotoxicity with strong mechanical and chemical membrane disruption. Multilayered MXene affects actin filaments and the associated stress-induced cytoskeleton changes. However, the MXene structure showed excellent cyto-compatibility with cell viabilities	33
Ti2AlC	HR-TEM	Structure		Cytotoxicity
Ti3AlC2	XRD	Structural property		ROS
M-Ti3C2Tx	Raman	Vibrational property		Morphology
D-Ti3C2Tx	AFM	Thickness
S-Ti3C2Tx	DLS	Zeta potential
Ti3AlC2	SEM	Morphology	Rice (Oryza sativa L.)	Germination	Ti3AlC2 nanosheets entered the leaf cells in the form of Ti ions and activated the immune system. It changes the structure and function of leaves, enhances the activity of antioxidant enzymes, damages cell membranes, reduces biomass, decreases chlorophyll, and inhibits plant growth	34
	TEM	Structure		Morphology
	HR-TEM	Structure		Structure
	EDS	Components		Function
	XRD	Crystal structure		Water content
				Activity of antioxidant enzymes
Ti3C2 MXene nanosheets	TEM	Thickness	Human embryonic kidney (HEK)-293 cell	Cytotoxicity	Ti3C2 MXene nanosheets for 3D bioprinting application showed high electrical conductivity, and improved mechanical characteristic high viability of most current bioinks	35
Ti3C2 MXene nanocomposite bioink	XRD	Presence of elements
	—	Swelling ratio, morphology, electrical conductivity, compression strength, rheological measurements
Ti3C2	SEM	Morphology	Spontaneously transformed aneuploid immortal keratinocyte cell (HaCaT), Human malignant melanoma (A375) cell	Viability	2D Ti3C2/PLL hybrids exhibited bactericidal properties and good biocompatibility in a relatively wide concentration range. The tested Ti3C2 MXene phase in both unmodified and surface-modified form did not show cytotoxicity	36
PLL	EDS	Elemental composition		Cellular cycle
Ti3C2/PLL	HRTEM	Morphology
	FFT	Layered reflection
	IFFT	Alternating layers
	DLS	Zeta potential, hydrodynamic diameters
	FTIR	Characteristic signals derived from bond vibrations
	—	Band intensity pattern, Isotherm of physical nitrogen sorption, bactericidal properties
Nb2C nanosheets	EDS	Elemental composition	Porcine cadaver skin, breast cancer cell from BALB/c nude mice (4T1)	Skin insertion and dissolution test	Microneedle (MN) system features a neat and complete macroscopic structure, sufficient compressive strength, excellent skin penetration and dissolution abilities, and high photothermal-ablation performance. The MNs exhibited no significant toxicity or adverse effects on the mice	37
Polyvinylpyrrolid one (PVP)	Electron microscope	Morphology		Cytotoxicity photothermal effect
Microneedle (PVP/Nb2C MN)	DLS	Size		Photothermal-ablation efficacy
	UV-vis-NIR spectrometer	UV-vis-NIR absorption spectra
	XPS	Presence of elements
	AFM	Thickness
	Zwick Materials Testing Machine	Compressive strength
	SEM	Morphology
GTA	FITR	Structure	Mouse fibroblast cell (L929 cell), mice, E. coli, S. aureus, methicillin-resistant S. aureus (MRSA)	Morphology	HPEM scaffolds displayed high-antibacterial activity against E. coli, S. aureus, and MRSA and could efficiently reach hemostasis and increase cell proliferation with negligible toxicity in vitro. HPEM significantly enhanced MRSA-infected wound healing via anti-infection, stimulating cell proliferation, an angiogenic process, up-regulating α-SMA and CD31, promoting granulation tissue formation, collagen deposition, vascular endothelial differentiation, and angiogenesis	38
PEI	SEM	Morphology		Viability
PGE	TEM	Structure		Skin recovery
HPEM	EDS	Elemental composition		Inflammation
HCHO	—	Viscosity, electronic conductivity, normal stress to the adhesive ability		Granulation tissue thickness
HPE				Collagen content
MXene@PDA				Antibacterial activity
				Hemostatic ability
Ti3C2Tx	TEM	Structure	Zebrafish embryo, human breast cancer cells (MCF7)	Mortality	No overall embryonic toxicity or teratogenicity effects for the nanocomposites even at high concentrations. For cancer therapy, efficient apoptosis and photothermal activity were revealed for Au/Fe3O4/MXene nanocomposites	39
Au/MXe ne	SEM	Morphology		Body deformity
Au/Fe3O 4/MXene	XRD	Presence of elements		Escoliosis
	VSM	Magnetization of ferromagnetic materials		Pigmentation
				Yolk edema
				Heart edema
				Movement defects
				Viability
2D Ti2NTx Mxene	DLS	Zeta potential, hydrodynamic diameters	Human malignant melanoma (A375) cell, spontaneously transformed aneuploid immortal keratinocyte cell (HaCaT), breast cancer cell (MCF-7), human female mammary glands (MCF-10A)	Cytotoxicity	2D Ti2NTx only toxic to cancer cells by generating intracellular reactive oxygen species	40
Ti2AlN	SEM	Morphology		ROS
	FFT	Layered reflection		Uptake
	HREM	Structure
	IFFT	Alternating layers
	EDX	Elemental composition
	UV-vis-NIR spectrometer	UV-vis-NIR absorption spectra
	FTIR	Characteristic signals derived from bond vibrations
AR-MX	SEM	Morphology	Human malignant melanoma (A375) cell, spontaneously transformed aneuploid immortal keratinocyte cell (HaCaT), breast cancer cell (MCF-7), human female mammary glands (MCF-10A)	Viability	Proposing that reactive oxygen species generation is one mechanism of toxicity and surface-Ti2O3 also recognized as a key factor inducing oxidative stress	41
SO-MX	UV-VIS	UV-vis-NIR absorption spectra		ROS
TO-MX	FTIR	Characteristic signals derived from bond vibrations
	XPS	Presence of elements
	DLS	Zeta potential
Ti3C2 QDs	TEM	Diameter	Human umbilical vein endothelial cells (HUVEC)	Cytotoxicity	Autophagic dysfunction leads to higher accumulation of pro-caspase 3	42
Nb2C QDs	AFM	Thickness		Inflammation
	HUVECs	Intracellular concentration		Autophagy
				Apoptosis
Ti3C2 Mxene	SEM	Thickness	Human malignant melanoma (A375) cell, spontaneously transformed aneuploid immortal keratinocyte cell (HaCaT), adenocarcinomic human alveolar basal epithelial cell (A549), Human fetal lung fibroblast cell (MRC-5)	Viability	Proposing reactive oxygen species generation as a possible mechanism of the cytotoxicity of 2D sheets of Ti3C2	9
	TEM	Structure		ROS
	HREM	Structure
	FFT	Layered reflection
	DLS	Zeta potential, particle size
	XPS	Presence of elements

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In the MXene studies, seven out of nine studies are biomedical application studies, breast cancer-related cells (MCF-7 and MCF-10A) were used in four studies,^37,39–41 skin cancer-related cells (HaCaT and A375) were used in four studies,^9,36,40,41 and kidney cells (HEK-293) and fibroblast cells (L929) were also used.^35,38 Alternative model species such as zebrafish embryos and bacteria have also been used.^38,39 For the toxicity endpoint, cell viability was tested in all studies, ROS measurement was performed in three studies,^9,40,41 and inflammatory response tests were performed in two studies.^38,42 Uptake, cellular cycle, photothermal effect, skin recovery, granulation tissue thickness, collagen content, autophagy, and apoptosis were also measured.^{36–38,40,42}

While extensive research has been conducted on the characteristics of MAX phases for diverse applications, investigations regarding their biocompatibility and toxicity remain scarce. A significant amount of research exploring biocompatibility is focused exclusively on MXenes. However, the lack of toxicity information on the MAX phase, the precursor of MXene, may hinder the development and utilization of various MXene-based products. Hence, this highlights the crucial importance of conducting comprehensive biocompatibility and toxicity studies on MAX phases, as they are lagging in research priority compared to their intrinsic value. In addition, gaining insight into the toxicity would greatly facilitate their utilization, particularly in the development of safe materials through an SbD approach. Therefore, in this study, toxicity screening assays were performed to ascertain a broader range of toxicity associated with MAX phases.

3.2. Physicochemical properties

MAX phases, Ti₂AlC and Ti₃AlC₂, were characterized by SEM, AFM, EDS, XPS, and XRD before the toxicity test to assess the physicochemical properties of nanomaterials in the test media (Fig. 1). Fig. 1(a) shows the SEM images of the Ti₂AlC and Ti₃AlC₂ particles before and after ultrasonication. Before ultrasonication, the sizes of the bulk Ti₂AlC and Ti₃AlC₂ particles ranged from a few micrometers to tens of micrometers. After ultrasonication, the Ti₂AlC and Ti₃AlC₂ particles were in the order of hundreds of nanometers in size. Fig. 1(b) and (c) show AFM images of the Ti₂AlC and Ti₃AlC₂ particles, respectively, to determine the thickness of the particles. The thickness of the particles ranged from 5 to 10 nm. Fig. 1(d) presents the EDS spectrum of the Ti₂AlC and Ti₃AlC₂ particles. Strong peaks corresponding to Ti, Al, and C were observed. XPS was conducted to identify the bonding on the surface of the MAX phase particles, as shown in Fig. 1(e). Ti₂AlC and Ti₃AlC₂ particles exhibited similar spectral features over the entire binding energy range. The binding energies of Ti 2p, Al 2p, and C 1s were 453, 71, and 281 eV, respectively.^43–45Fig. 1(f) shows the measured XRD pattern of the Ti₂AlC and Ti₃AlC₂ samples. The Ti₂AlC XRD pattern exhibited nine XRD peaks at 2θ = 12.95° (002), 26.1° (004), 33.9° (100), 39.45° (103), 43.35° (104), 53.1° (106), 60.75° (110), 71.75° (109), and 74.95° (116).⁴⁶ The Ti₃AlC₂ sample showed 14 peaks at 2θ = 9.54° (002), 19.14° (004), 34.02° (101), 36.79° (103), 39° (104), 41.74° (105), 44.96° (106), 48.5° (107), 52.56° (108), 56.42° (109), 60.2° (110), 65.48° (1011), 70.4° (2021), and 74.12° (2024).^46,47 The highly intense, sharp (002) peak indicates that the MAX phase particles had a high degree of crystallinity. The physicochemical properties of GO were analyzed in a previous study,¹⁴ and the methods and results are provided as ESI† (Table S1).

Fig. 1 (a) Measured SEM images of bulk and sonicated Ti₂AlC and Ti₃AlC₂ particles. Measured AFM images of (b) Ti₂AlC particles and (c) Ti₃AlC₂ particles. Measured (d) EDS spectrum and (e) XPS spectra, and (f) XRD patterns of Ti₂AlC and Ti₃AlC₂ particles.

The dispersion stability of the MAX phases (Ti₂AlC and Ti₃AlC₂) was determined by DLS in the test media for cytotoxicity and ecotoxicity (Fig. 2). DLS was conducted immediately after spiking (time 0) and 24 h after spiking. The measured values of the absolute zeta potential were small (<−10 mV) for MAX phases in the tested media, except for Ti₃AlC₂, at 0 h after spiking. The DLS results of each medium collectively suggest that the colloidal dispersion stability of MAX phases and GO showed similar fluctuations over time in all media tested, except for 0 h Ti₃AlC₂.

Fig. 2 (a) Zeta potential and (b) hydrodynamic diameter of MAX phases (Ti₂AlC and Ti₃AlC₂) in the test media for cytotoxicity and ecotoxicity. Graphene oxide (GO) was used as a reference material.

3.3. Cytotoxicity of MAX phases

The effects of the two MAX phases on human health were first screened by investigating their cytotoxicity using BEAS-2B cells at various time points (2, 4, 6, 8, 12, and 24 h) (Fig. 3). The cytotoxicity demonstrated a significant time-dependent increase within the first 6 h but showed less pronounced time dependency thereafter. Ti₃AlC₂ induced more severe cytotoxicity than Ti₂AlC in the first 6 h, whereas Ti₂AlC was more cytotoxic than Ti₃AlC₂ at 24 h. Overall, MAX phases and GO induced cytotoxicity within the range of 10–20%, and there were no significant differences in cytotoxicity among them (Fig. 3).

Fig. 3 Cytotoxicity of MAX phases (Ti₂AlC and Ti₃AlC₂) to BEAS-2B cells. Graphene oxide (GO) was used as a reference material. The cells were exposed to MAX phases and GO at 1 mg L⁻¹ for different time points and % cytotoxicity determined by RealTime-Glo™ MT cell viability assay in DMEM F/12 medium. The experiments were performed for three individual replications, each with two technical replicates. The data are presented as the mean ± standard error of the mean.

The cytotoxicity of MAX phases and MXenes has been previously investigated. According to Scheibe et al., Ti₂AlC and Ti₃AlC₂ exhibited cytotoxicity in normal human fibroblast (MSU1.1) cells at concentrations equal to or greater than 50 μg mL⁻¹.³³ Gu et al. confirmed cytotoxicity in HUVECs cells at concentrations equal to or greater than 50 mg L⁻¹ for Ti₃C₂ MXene.⁴² Similarly, Rozmysłowska-Wojciechowska et al. observed cytotoxicity in HaCaT and A375 cells exposed to Ti₃C₂ MXenes at concentrations of 500 mg L⁻¹.³⁸ In this study, we exposed MAX phases at a concentration of 1 mg L⁻¹, resulting in approximately 20% cytotoxicity. Therefore, this concentration has been established as suitable for conducting subsequent toxicity investigations.

3.4. Genotoxic potential of MAX phases

Oxidative stress is known to be an initial toxic effect caused by exposure to various nanomaterials,^48,49 which can lead to subsequent toxic effects such as inflammation, DNA damage, and more. In particular, it is well-established as an upstream event in genotoxicity.^50,51 As evident from the literature analysis (Table 2), most toxicity studies on MAX phases and MXenes have measured ROS and oxidative stress. Hence, this study aims to investigate the potential genotoxic capability of MAX phases.

A tiered approach was used, combining comet assay for DNA double-strand breaks and previously established C. elegans functional genetic tool⁵² for DNA repair capacity. The direct DNA damage in BEAS-2B cells was then investigated using alkaline comet assays (Fig. 4(a)). The olive tail moment, a DNA strand break marker, increased significantly in BEAS-2B cells exposed to both MAX phases (Ti₂AlC and Ti₃AlC₂). This result collectively suggested that MAX phases had genotoxic potential in BEAS-2B cells, and their intensity was comparable to that of GO.

Fig. 4 DNA damage and inflammation of MAX phases. Graphene oxide (GO) was used as a reference material. (a) DNA damage measured in MAX phases (Ti₂AlC and Ti₃AlC₂) and GO exposed BEAS-2B cells using the comet assay. MAX phases and GO were exposed at 1 mg L⁻¹ for 24 h. Pb²⁺ was used as the positive control. (b) Response of C. elegans DNA repair enzyme gene mutants to MAX phases (Ti₂AlC and Ti₃AlC₂) and GO exposure. MAX phases and GO were exposed at 1 mg L⁻¹ for 72 h. (c) Expression of inflammation marker IL-6 in MAX phases and GO exposed BEAS-2B cells. MAX phases and GO were exposed at 1 mg L⁻¹ for 24 h. All results are shown as the mean ± standard error of the mean. *p < 0.05, **p < 0.01 compared to the control (one-way analysis of variance).

The DNA repair pathways in C. elegans are evolutionarily conserved in humans.^53–55 The response of the DNA repair enzyme gene (nth-1 and ndx-4) loss-of-function mutants was compared with that of wild-type N2 (Fig. 4(b)). In nth-1 and ndx-4 mutants, increased toxicity was observed after GO exposure, suggesting that the BER pathway was required for defense against GO. Therefore, GO might cause genotoxicity, which is consistent with previous studies.^14,56,57 The nth-1 and ndx-4 are both involved in the base-excision repair (BER) of DNA damage, which is often caused by oxidative stress.^58,59nth-1 is a C. elegans homolog of human endonuclease III (NTH) and a well-conserved DNA glycosylase that removes oxidized pyrimidine bases,⁶⁰ and ndx-4 has a MutT-type function that can hydrolyze 8-oxodGTP.⁶¹ The toxicity of the BER-defective mutants increased with exposure to the MAX phases, particularly with Ti₃AlC₂, therefore, the toxicity mechanism of MAX phases might be involved in the BER pathway. Although the response of Ti₃AlC₂ was slightly higher than that of Ti₂AlC, the difference was not statistically significant. Both MAX phases showed lower genotoxic potential than GO in C. elegans.

In order to elucidate the underlying mechanism of genotoxicity, inflammation was assessed as it facilitates DNA repair in response to DNA double-strand breaks.^62,63 IL-6, a proinflammatory cytokine marker was investigated in BEAS-2B cells exposed to the MAX phases (Fig. 4(c)). A statistically significant increase in the IL-6 level was observed in the Ti₂AlC exposure group; however, no significant difference was found in the Ti₃AlC₂ group. IL-6, a cytokine associated with oxidative stress,⁶⁴ has been reported to be linked to genotoxicity in various nanomaterials such as graphene and carbon black.^65,66 Therefore, we hypothesized that the genotoxicity induced by MAX phases in BEAS-2B cells could be attributed to augmented ROS production,^9,33,40,41 as well as inflammatory responses, leading to secondary genotoxic effects.⁶⁶ A wide range of molecular and biochemical stress markers should be investigated to gain insight into the consequences of DNA damage at the cellular level.

3.5. Ecotoxicity of MAX phases

Most studies investigating the toxicity of MAX phases have primarily focused on human health, while there is a notable dearth of research on ecotoxicity based on our current knowledge (Table 2). Considering the potential for environmental exposure to MAX phases, it is imperative to conduct studies addressing ecotoxicity. To gain insight into the potential environmental hazards of MAX phases, a battery assay was performed using representative soil and aquatic model organisms, C. elegans and zebrafish (Fig. 5). Exposure to MAX phases did not cause any significant toxicity in C. elegans (Fig. 5(a)) and zebrafish embryo (Fig. 5(b)) at a concentration of 1 mg L⁻¹.

Fig. 5 Number of offspring (a) in MAX phases exposed C. elegans. (b) Mortality of zebrafish embryo exposed to MAX phases. Graphene oxide (GO) was used as a reference material. MAX phases and GO were exposed for 96 h at 1 mg L⁻¹.

MAX phases might induce genotoxicity and cytotoxicity in a human cell line, but not at the whole organism level. It is important to note that these results should not be interpreted as MAX phases not causing ecotoxicity, as toxicity generally occurs at lower concentrations at the molecular level than at the individual level.⁶⁷ One possible explanation for these results could be the differences in the uptake of the MAX phases, resulting in varying concentrations within each biological system. In our study, both cells and model organisms were exposed indirectly through liquid media. However, zebrafish embryos benefit from the protection of the chorion, and C. elegans necessitates ingestion for material to enter their bodies. Although our research did not directly confirm the uptake, given the nano-sized nature of MAX phase particles, it is reasonable to assume significant uptake. Comparing the sizes of MAX phases (less than 1 μm for Ti₂AlC and around 4 μm for Ti₃AlC₂) to those of the reference material, GO with a hydrodynamic diameter of 20–40 μm, suggests that MAX phases are also likely to be internalized by organisms. Previous studies have demonstrated GO uptake by C. elegans⁶⁸ and its ability to traverse the chorion of zebrafish embryos.^69,70

Nevertheless, compared to cell-based assays, it is reasonable to assume that lower concentrations of the MAX phase reach the cell interior, potentially explaining the absence of toxicity at the organism level. Therefore, comprehensive research and understanding of the uptake and kinetics of MAX phases within living organisms are essential. Of course, one could interpret the absence of toxicity as a simple interspecies difference between humans and ecological species. Additionally, this study focused on short-term exposure effects, but given the observed toxicity at the cellular level, further investigations are necessary to examine the effects of long-term exposure at the organism level.

4. Considerations for SbD implementation

The expansion of MAX phase technology has led to a wide range of applications for these materials, owing to their unique metallic and ceramic properties. However, novel applications of MAX phases and its 2-dimensional derivative MXenes may introduce new hazards. This study has focused on assessing the potential hazards and toxicity mechanisms of two MAX phases, Ti₂AlC and Ti₃AlC₂, for environmental health and safety assessments, highlighting their non-negligible hazardous potential compared to GO. Although generalization of the toxicity profile of the MAX phase should be avoided, these results suggest that significant attention should be paid to the mass production and application of the MAX phase. Implementing the SbD concept during the R&D or design phase of MAX phases and MXenes would proactively address these risks and contribute to sustainable development.

The first step of SbD involves utilizing less harmful substances based on their known toxicity.¹⁷ In our study, the results revealed that Ti₃AlC₂ exhibited lower level of cytotoxicity, DNA damage, and inflammatory response (IL-6) at 24 h in comparison to Ti₂AlC. It is essential to recognize that nanomaterials such as MAX phases can exhibit variations in toxicity due to factors like composition, size, aspect ratio, and other physicochemical properties. However, there is a notable lack of comprehensive studies regarding the toxicity of MAX phases, making it difficult to identify specific factors that influence toxicity. The two materials examined in our study share very similar physicochemical properties. Nonetheless, Ti₃AlC₂ exhibited a hydrodynamic diameter approximately four times larger than Ti₂AlC and a thinner profile, resulting in a higher aspect ratio. Previous studies have indicated that the relationship between toxicity and aspect ratio may vary depending on experimental conditions or nanomaterial type.⁷¹ Under the specific experimental conditions of our study, higher aspect ratios in MAX phases were associated with reduced toxicity. However, as this interpretation is based on a single characteristic, it would be prudent to draw comprehensive conclusions by considering a broader range of characteristics. Therefore, continued efforts should be made to compare the toxicity of MAX phases with various compositions and physicochemical properties using well-established experimental methods to identify safer materials with similar functionalities. Such research will serve as a valuable foundation for supporting the implementation of the SbD approach.

Key physicochemical properties influencing the toxicity of nanomaterials include lateral size, surface structure, functionalization, charge, and impurities.^11,72,73 Changes in these properties may alter the toxicity, necessitating additional safety evaluations. To avoid the challenges of individually testing each change, it is crucial to establish the relationship between these properties and toxicity, enabling toxicity prediction without extensive experimental testing. Many studies have explored this relationship, leading to toxicity prediction models for nanomaterials.^74–77 Developing an in silico model optimized for MAX phases based on these studies could significantly enhance the development of safer materials. Additional research in this direction could further advance the SbD approach for MAX phases.

Exposure is also a critical factor influencing the toxicity of nanomaterials. In our study, a concentration of 1 mg L⁻¹ was used, which is relatively low compared to previous studies.^33,38 As environmental concentration data for MAX phases are currently unavailable, we adopted this concentration based on a study setting the environmental concentration of GO.⁷⁸ Thus, our experiment simulated low-exposure scenarios in the environment rather than high-exposure scenarios in an occupational accident. Since MAX phases are still in the development stage, the current exposure to MAX phases mainly occurs through inhalation in the workplace, so future research should focus on inhalation exposure and toxicity. For in vitro studies, an air–liquid interface system may be employed.⁷⁹ In the long term, it is necessary to understand the potential exposure of MAX phases to humans and ecological species and determine the concentrations encountered during the manufacturing process and use in terms of a life cycle assessment.⁸⁰

5. Conclusions

In this study, two types of MAX phase materials (Ti₂AlC and Ti₃AlC₂) were characterized, and their cytotoxicity, genotoxicity, and ecotoxicity were investigated to gain insight into their potential toxicity, thus enabling the implementation of the SbD approach. Our study highlights the importance of assessing the potential hazards and toxicity mechanisms of MAX phases in the context of human and environmental safety. These findings provide valuable insights and a basis for implementing SbD approaches in the development and application of MAX phases for safer and environmentally conscious nanotechnology advancements.

Author contributions

Jaeseong Jeong: investigation, visualization, writing – original draft, writing – reviewing and editing. Su-Yong Bae: investigation, formal analysis, validation, writing – original draft. Jinho Lee: methodology, visualization, writing – original draft. Suh-Young Kwon: formal analysis, writing – original draft. Ju Han Lee: resources, writing – reviewing and editing. Jinhee Choi: supervision, conceptualization, writing – original draft, writing – reviewing and editing.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the Korean Ministry of Environment under the ‘Environmental Health R&D Program’ (2021003310005) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2021R1A5A1032937).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3en00528c

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