2-D transition metal trichalcophosphogenide FePS₃ against multi-drug resistant microbial infections

Abstract

Antimicrobial resistance (AMR) is a significant concern to society as it threatens the effectiveness of antibiotics and leads to increased morbidity and mortality rates. Innovative approaches are urgently required to address this challenge. Among promising solutions, two dimensional (2-D) nanomaterials with layered crystal structures have emerged as potent antimicrobial agents owing to their unique physicochemical properties. This antimicrobial activity is largely attributed to their high surface area, which allows for efficient interaction with microbial cell membranes, leading to physical disruption or oxidative stress through the generation of reactive oxygen species (ROS). The latter mechanism is particularly noteworthy as it involves the degradation of these nanomaterials under specific conditions, releasing ROS that can effectively kill bacteria and other pathogens without harming human cells. This study explores the antimicrobial properties of a novel biodegradable nanomaterial based on 2-D transition metal trichalcogenides, FePS₃, as a potential solution to drug-resistant microbes. Our findings indicate that FePS₃ is an exceptionally effective antimicrobial agent with over 99.9% elimination of various bacterial strains. Crucially, it exhibits no cytotoxic effects on mammalian cells, underscoring the potential for safe biomedical application. The primary mechanism driving the antimicrobial efficacy of FePS₃ is the release of ROS during biodegradation. ROS has a crucial role in neutralizing bacterial cells, conferring significant antipathogenic properties to this compound. The unique combination of high antimicrobial activity, biocompatibility, and biodegradability makes FePS₃ a promising candidate for developing new antimicrobial strategies. This research contributes to the increasing body of evidence supporting the use of 2-D nanomaterials in addressing the global challenge of AMR, offering a potential pathway for the development of advanced, effective, and safe antimicrobial agents.

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1. Introduction

The global health challenge posed by microbial resistance to antibiotics is substantial, leading to elevated rates of sickness and mortality on a worldwide scale.^1–4 Antimicrobial resistance (AMR) refers to the capacity of microorganisms, including bacteria, viruses, parasites, and fungi, to endure and withstand the impact of medications that were previously effective in addressing infections.^5,6 This phenomenon is driven by several factors, including the misuse and overuse of antimicrobial drugs in human and animal health as well as in agriculture.^7–9 Resistance is a natural response of microorganisms to selective pressure imposed by antimicrobial drugs.^10–12 These consequences are far-reaching and have the potential to undermine the effectiveness of modern medicine. The Australian Centre for disease control estimated that the annual impact of antimicrobial resistance on the Australian economy would lie between A$142 billion and A$283 billion.¹³ Moreover, an increasing trend has been observed in the prescription of first-line antibiotics by general practitioners.¹⁴ The six major pathogens responsible for more than 80% of deaths annually include; Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and, Staphylococcus aureus.^15–17 AMR is predicted to cause more than 10 million fatalities by 2050.¹⁸ If left unaddressed, common infections can become untreatable, leading to prolonged illness, increased healthcare costs, and higher mortality rates. In addition to its direct impact on human health, AMR has economic and societal impacts, threatening food security, animal health, and sustainable development.^19–21 Furthermore, there has been a steady decline in the discovery of newer drugs capable of killing these microbes, leading to a shortage of effective antimicrobials in the pharmaceutical sector.^22,23 This decrease can be attributed to various factors such as regulatory obstacles, high research and development expenses, and the absence of financial incentives for pharmaceutical companies.²⁴ Additionally, there is a lack of knowledge among healthcare professionals and the public regarding the proper use of antibiotics, which results in overprescription and misuse, exacerbating the issue.²⁵

Being a complex and urgent global health challenge, AMR requires a multifaceted approach that includes thorough monitoring and investigation, advocating the appropriate utilization of antimicrobial medications in healthcare and farming, and creating novel medications and treatment approaches to remain ahead of resistant microorganisms.²⁶

Due to the inadvertent reduction in the discovery of newer first-line antimicrobials, there has been a notable focus on the development of next-generation antimicrobial therapeutics in response to the rise in antibiotic-resistant bacteria, and the need for novel strategies to combat infectious diseases has become crucial.^27–30 This has led to a surge in research on the development of a new generation of nanomaterial-based antimicrobials.^31–33 Nanomaterials have emerged as promising tools in this battle, offering unique mechanisms for counteracting antimicrobial resistance. They are a group of materials whose dimensions are of the order of 100 nm or less,³⁴ and have garnered substantial interest owing to their ability to induce oxidative stress, release metal ions, and exhibit non-oxidative mechanisms as well as synergistic effects that combine these mechanisms.^35,36 These properties have paved the way for the formulation of a new generation of nanomaterial-based antimicrobials, presenting a potential solution to the increasing ineffectiveness of traditional antimicrobials against pathogens. Consequently, there has been a significant increase in the development of nanomaterial-based surface antimicrobials to effectively disrupt bacterial and fungal biofilms.³⁷

Two-dimensional (2-D) transition metal phosphorus trichalcogenides (2D-TMPX's), a class of compounds that contain transition metals (such as molybdenum or titanium), have shown potential for addressing drug-resistant bacterial infections.³⁸ These materials belong to the family of transition metal dichalcogenides (TMDs), which have been extensively studied owing to their remarkable electronic, optical, and catalytic characteristics.^39–43 They have an empirical formula of MPX₃, where M is a transition metal, P is phosphorus, and X is the chalcogen atom. These include MnPS₃ and FePS₃. The crystal structure of metal–phosphorus trichalcogenides typically consists of a phosphorus layer sandwiched between two chalcogen layers (sulphur, selenium, or tellurium), forming a two-dimensional honeycomb-like arrangement. This layered structure, held together by van der Waals interactions, allows easy exfoliation and the creation of ultrathin nanosheets with a high surface-to-volume ratio. Some TMPX's are biodegradable under atmospheric conditions, leading to the sustained release of reactive oxygen species (ROS). Ramos et al., (2021) found that the biodegradation of FePS₃ was influenced by environmental factors such as pH and temperature, which can impact the antimicrobial dynamics of the material.⁴⁴

In this work, we demonstrate the potential antimicrobial activity of few-layered FePS₃ nanoflakes in nanogram concentrations. According to our findings, FePS₃ has exceptional antimicrobial properties in nanogram quantities against a diverse range of microbes, including: Pseudomonas aeruginosa (P. aeruginosa), methicillin-resistant Staphylococcus aureus (MRSA), and the fungal strain, Candida albicans (C. albicans). Furthermore, the compound exhibited lower toxicity levels on mammalian cells, and no hemolysis upon interactions with the blood cells. The confocal laser scanning microscope (CLSM) analysis showed that the nanoflakes produced a significant reduction in both higher and lower bacterial population densities, with 99.99% elimination of drug-resistant bacterial strains. This is attributed to the ability of FePS₃ to self-degrade upon interaction with ambient oxygen and light to release ROS, as confirmed by molecular dynamics (MD) simulations. This study provides an in-depth understanding of the antipathogenic properties and the mechanism of action of the nanomaterial. This will further aid in the development of nanomaterial-based treatment regimens for drug-resistant bacterial infections in the future.

2. Results

2.1. Characterization of FePS₃ flakes

FePS₃ belongs to the transition metal phosphorus trichalcogenides family. Its structure is characterized by a lamellar arrangement with strong intralayer bonding within the layers, and the adjacent layers are held together by weak van der Waals interactions. Each layer comprises phosphorus atoms bonded to six sulphur atoms, which form trigonal prismatic coordination, with the iron atoms sandwiched between the layers of the phosphorus–sulphur networks (Fig. S1†). This compound exhibits semiconducting behaviour with an absorption edge in the near-infrared spectrum and displays Ising-like antiferromagnetism.⁴⁵ A few layers of FePS₃ (which is mainly composed of 10–30 stacks of the compound weakly held together by van der Waals forces) are achieved by mechanically exfoliating the bulk compound using the Scotch-tape method.⁴⁶Fig. 1A and S2A† depicts an SEM (scanning electron microscopy) image of the FePS₃ flake. Electron dispersive X-ray spectroscopy was used to analyse and map the distinct energy peaks associated with the elements, with iron and sulphur (Fig. 1B and D) predominantly present in the compound.⁴⁷ Phosphorus has a lower peak, probably due to its degradative property under ambient conditions (Fig. 1C). High resolution transmission electron microscopy (HR-TEM) images were acquired to determine the morphology of exfoliated nanoflakes.⁴⁸ Defect-free crystalline nanoflakes were observed in the HR-TEM image (Fig. 1E and S2B†). ATR-FTIR analysis of the mechanically exfoliated FePS₃ nanoparticles is shown in Fig. 1F. The infrared spectrum of the synthesized nanoflakes was in the range 500–4000 cm⁻¹. The intense band at 608 cm⁻¹ (607–622 cm⁻¹) is associated with the stretching vibrations of the Fe–S bonds. A strong, broad stretch of the Si–C bond was located at 736 cm⁻¹. Finally, the phosphate bonds showed an asymmetric stretch in the range of 1100–1300 cm⁻¹. AFM imaging of mechanically exfoliated nanoparticles helps visualize variations in flake distribution and sizes.⁴⁹Fig. 1G shows the average size and height profiles of the flakes in a heterogeneous batch of nanoparticles. The average dimensions of the flakes range from 1–5 μm, with smaller fragments being less than 50 nm. Additionally, the average thickness of the flakes ranged from to 5–65 nm, with a higher proportion of them being to 25–30 nm (Fig. 1H). The average lateral size of the flakes was also consistent, with a larger proportion of them ranging from 2–4 μm (Fig. 1I). We estimated the average mass of mechanically exfoliated FePS₃ deposited on substrate using AFM images. The average surface coverage percentage (A_FePS3) was calculated by applying a height threshold cutoff to isolate areas covered by nanoflakes from a series of 30 μm² AFM scans. The mean flake height (H_FePS3) and surface coverage were then used to calculate the flake volume per cm². By applying the material's density (5.14 g cm⁻³), we derived the mass per unit area of the deposited FePS₃. This method yielded an approximate flake deposition of 816.74 ± 12.97 ng cm⁻²via mechanical exfoliation.

Fig. 1 General characterization of FePS₃ nanoflakes (A) SEM image of exfoliated FePS₃ flakes. (B) EDS map of iron, (C) phosphorus, and (D) sulphur. (E) HR-TEM image of a larger exfoliated flake. (F) ATR FT-IR spectra of FePS₃ flakes showing peaks corresponding to Fe–S, Si–C, and phosphate bonds. (G) AFM image of the exfoliated particles of FePS₃ flakes, illustrating the morphology of mechanically exfoliated flakes, (H) average thickness and (I) lateral width of the flakes. The observed flake dimensions highlight their suitability for surface interactions in antimicrobial applications, where uniform morphology can enhance interface-bound ROS generation and microbial contact.

2.2 Assessing the biodegradability of the flakes

To further understand the nature of its degradability, the exfoliated flakes were subjected to AFM and XPS analyses after exposure to atmospheric conditions for seven days. Fig. 2A shows the morphological differences between the flakes during AFM imaging. AFM images captured on day 1 revealed that the FePS₃ flakes were intact with well-defined edges and a relatively smooth surface morphology. The flake thickness measurements corresponded closely to the expected values for pristine FePS₃, indicating a minimal initial degradation or surface irregularities. By day 7, a significant degradation was observed in the AFM images. The edges of the FePS₃ flakes exhibited a noticeable breakdown, with fragment detachment and the edges becoming increasingly irregular. The surface roughness increased markedly, indicating a substantial degradation. This edge degradation suggests that the flakes are particularly susceptible to atmospheric conditions, likely because of the higher reactivity of the edge atoms compared to those in the bulk.⁵⁰ The XPS spectra obtained on day 1 showed characteristic peaks for phosphorus in FePS₃, with binding energies corresponding to the expected oxidation states of phosphorus in the pristine sample. By day 7, there was a significant shift in the phosphorus peaks. The P 2p spectrum indicates the presence of oxidized phosphorus species, as evidenced by the emergence of new peaks at higher binding energies associated with phosphate. This shift confirms that the phosphorus atoms in the FePS₃ structure oxidize over time when exposed to atmospheric conditions.⁵¹ The Fe 2p spectra exhibited notable changes between days one and seven. Initially, the Fe 2p^3/2 and Fe 2p^1/2 peaks were consistent with the expected chemical state of iron within the FePS₃ structure. By day 7, there was an alteration in the Fe 2p spectrum, with new features and a shift in the binding energy of the primary Fe 2p^3/2 peak. This suggests a change in the chemical state of iron, likely due to the formation of iron oxides or hydroxides. Unfortunately, due to the multiplet splitting, the analysis of the iron peaks becomes complex (Fig. S3A†). The sulphur peaks did not exhibit a drastic shift (Fig. S3B†). Additionally, the SEM image of a flake placed in atmospheric conditions for 24 h depict the breakdown of the flake, with the formation of phosphorus oxides, as seen on the EDS results (Fig. S4†).

Fig. 2 Degradative property of nanoparticles. (A) AFM images of FePS₃ nanoflakes showing edgewise degradation of the surface (A) day 1 and (B) day 7. (C) XPS of exfoliated flakes showing peaks in the phosphorus components.

2.3. Antimicrobial properties of mechanically exfoliated FePS₃

The initial assessment of antimicrobial capabilities of FePS₃ nanoflakes was carried out by measuring the colony count of a range of multidrug-resistant microbial strains, such as MRSA, drug resistant variant of Pseudomonas aeruginosa, and Candida albicans. The percentage viability of the cells was determined and plotted as a bar chart. 99.99% (log 4 reduction), 99.9% (log 3 reduction) and 50% (log 0.3 reduction) elimination were observed with the flakes against MRSA, Pseudomonas aeruginosa and Candida albicans respectively (Fig. 3). Fig. S5† presents the antimicrobial activity of the positive controls (tetracycline and fluconazole), which confirms the effective inhibition of the tested microbial strains, particularly against MRSA and P. aeruginosa.

Fig. 3 Preliminary antimicrobial assay of FePS₃ performed against methicillin resistant Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, (A) corresponding agar plate images, (B) colony enumeration after serial dilution of the microbes. Values are mean ± SEM. n = 3. (B) Statistical analysis by paired t-test, ***p < 0.0001.

Following a preliminary investigation, the antimicrobial properties of FePS₃ were further determined against an increasing series of microbial optical densities using confocal scanning microscopy (CLSM). The bacterial (MRSA and P. aeruginosa) and fungal (C. albicans) samples of 0.01, 0.1, and 0.5 O.D. (optical density) were exposed to the flakes for 24 h. Differential staining was used to distinguish between live and dead cells in the samples.⁵² Viable cells stained green (SYTO 9) and dead cells stained red (propidium iodide). SYTO 9 is a nucleic acid binding stain that attaches to the viable nucleic acids present on the cell membrane, while propidium iodide binds to the damaged nucleic acids as well. Fig. 4A and B show confocal images of bacterial samples after exposure to the nanoflakes, using the live/dead assay. The control represents untreated samples. The percent of live to dead bacteria were enumerated and plotted on the bar graph shown in Fig. 4C and D. The CLSM image and colony enumeration of the fungal strain is shown in Fig. S6.† Similar to the preliminary tests, the nanoflakes had a higher percentage of cell death with the bacterial strains, with almost 99% of elimination seen at all densities. The fungal cells, on the other hand, only showed an inhibition of 30%.

Fig. 4 Antimicrobial effects of FePS₃ nanoparticles against bacterial populations at various optical densities. (A) Confocal laser scanning microscopy (CLSM) images showing the effect of FePS₃ nanoparticles on MRSA (methicillin-resistant Staphylococcus aureus) at three different optical densities (O.D.₆₀₀ = 0.05, 0.1, and 0.5) compared to untreated control groups. Green fluorescence represents live cells, and red fluorescence indicates dead cells stained with propidium iodide (PI). (B) CLSM images depicting the effect of FePS₃ nanoparticles on Pseudomonas aeruginosa across the same optical densities as in (A). (C) Quantification of cell death in MRSA following treatment with FePS₃, expressed as a percentage of dead cells relative to total cells, across the three optical densities. (D) Quantification of cell death in P. aeruginosa after FePS₃ treatment, similarly, expressed as a percentage. (E) & (F) Bacterial surface density (number of bacteria per μm²) afetr 24 hours of MRSA and P. aeruginosa at different optical densities in both control and FePS₃-treated groups, showing the reduction in bacterial attachment and growth after treatment. Values are mean ± SEM. n = 3. (B) Statistical analysis by one-way ANOVA, ***p < 0.0001.

To assess the antimicrobial and antifouling properties of the tested compound, the number of bacteria per square micrometre (bacteria per μm²) was determined using confocal microscopy. The total number of bacterial cells observed in each confocal image was counted and normalized to the image area by dividing by 10 000 (as each image represents a 100 × 100 μm area). Fig. 4E and F illustrate the bacterial density (bacteria per μm²) of MRSA and P. aeruginosa under varying optical densities (O.D 0.05, 0.1, and 0.5). The results demonstrate a significant reduction in bacterial density across all tested optical densities, after the addition of FePS₃. This reduction was consistent for both MRSA and P. aeruginosa, indicating that the compound effectively inhibits bacterial growth and prevents the undesirable attachment of bacterial cells and their by-products on surfaces.

2.4 Cytotoxicity of FePS₃ nanoflakes

Cell viability was evaluated against human immortalized keratinocyte (HaCaT) cells for 24 and 48 h using the same concentration of mechanically exfoliated FePS₃ flakes.⁵³ The flakes were placed in triplicate wells of a 24-well plate and HaCaT cells were added. The cells were then incubated, and their viability was determined using a resazurin metabolic assay. The fluorescence of the samples was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability was determined by the ability of live cells to convert a non-fluorescent resazurin dye into a fluorescent resorufin derivative. Mammalian cells showed no morphological changes after encountering the flakes, as shown in Fig. 5A and B. Fig. 5C shows the normalised viability of the cells after incubation with exfoliated flakes for 24 and 48 h. Additionally, a spectrophotometric reading was taken for the bare well plate and a well plate functionalised with FePS₃ in PBS. This reading did not show a significant difference in the adsorbance (Fig. S7†), which shows that the addition of FePS₃ did not interfere with the viability measurement conducted for the mammalian cells. The hemolysis assay was also performed against red blood cells (RBCs). FePS₃ flakes did not induce hemolysis when tested against blood cells (Fig. S8†).

Fig. 5 Cytotoxicity assay against HaCaT cells after 24 h and 48 h. Light microscopy images of the (A) control and (B) FePS₃ treated samples. (C) Normalised viability of the cells after treatment with few-layered FePS₃ flakes. Error bars represent the standard deviation from three independent experiments.

2.5 Antimicrobial mechanism of FePS₃ flakes

FePS₃ breaks down under atmospheric conditions, which can lead to the generation of ROS under ambient conditions and play a crucial role in its antimicrobial action. ROS can cause oxidative damage to the microbial cell membranes, proteins, and DNA, leading to cell death. To elucidate the antimicrobial mechanism, FePS₃ was tested for ROS generation. Singlet oxygen species (¹O₂), hydroxyl radicals (OH˙), and superoxide radicals (O₂˙⁻), are some of the ROS species involved. Specific dyes, including methylene blue (singlet oxygen species), xanthine oxidase (superoxide radicals), and horse radish peroxidase (hydroxyl radicals), were used to identify the types of species produced.

After 2 h of exposure to atmospheric conditions, detectable changes in absorbance were observed for all three dyes, indicating the presence of ROS. Notably, singlet oxygen exhibited the highest variation (Fig. 6). The higher variation in singlet oxygen suggests that it may be the dominant species contributing to the antimicrobial activity. However, the exact origin of ROS remains unclear and requires further investigation.

Fig. 6 ROS radical generation of FePS₃ nanoflakes. (A) O₂˙⁻, (B) ¹O₂ and (C) OH˙. UV absorption change after 2 h of flake exposure to atmospheric conditions. (D) Statistical analysis by one-way ANOVA, **p < 0.001 ***p < 0.0001. Error bars represent the standard deviation from three independent experiments. Values are mean ± SEM. n = 3.

2.6 Computational investigation of the antimicrobial mechanism of FePS₃ flakes

To better understand the mechanism of ROS generation by FePS₃, ab initio molecular dynamics (AIMD) simulations were performed on pristine and single defect (i.e., one S atom removed from the surface) unit cells in the presence of water and O₂. Similar to our previous study of few-layer black phosphorus (BP),⁵⁴ no reaction was observed with the pristine FePS₃, while the single-defect system reacted spontaneously with O₂ (Fig. 7). Interestingly, the addition of O₂ to the defect site significantly perturbs the positions of all S atoms in the unit cell, even those not directly adjacent to the defect, with one O atom interacting with the P atom at the defect site and the second O atom interacting with the P and two Fe atoms directly beneath it. The –P–O moiety at the surface is similar to that of the oxidized BP and can therefore reduce dissolved O₂ to superoxide in a similar way, while the production of singlet oxygen likely occurs during the decomposition process.⁵⁵

Fig. 7 Optimized geometry of FePS₃. (A) Original structure, (B) single defect created by removing one S atom from the surface, (C) oxidized defect surface.

3. Discussion

Nanotechnology, such as the application of nanoparticles, low-dimensional materials, and surface coatings, have emerged as being applicable as next-generation antimicrobial technologies.^56–59 The rationale behind such studies has been to investigate alternative therapies to conventional treatment methods, such as antibiotics.^32,60,61 Numerous avenues have been explored, including the study of nano-particles and nano-materials, as new antimicrobial therapeutic measures.^62–71 Two-dimensional and low-dimensional materials have received considerable attention as potential antimicrobial agents. These materials have included graphene,⁷² graphene oxide,^72–79 molybdenum disulphide,^74,80 and black phosphorus (BP).^54,81–86 A more thorough summary of antimicrobial nanomaterials – nanoparticles and nanoflakes – is included as Table S1 in the ESI† of this article, including our study. Notably, BP-based therapeutics^{81–83,87,88} exploit the natural degradation of the material as the antimicrobial mechanism, whereby ROS is generated and damages cellular components – DNA, proteins, and lipids – leading to oxidative stress and cell death.^89–92 In chemical terms, BP-based material will produce significant quantities of ˙OH, ˙O₂⁻, and H₂O₂, under ambient conditions in air and in solution,^92–96 where the precise concentration of ROS increases as a function of time and the initial base material concentration.^89–96 Although the precise reasons are still unclear, ROS appear to be well tolerated by mammalian cells,^54,84 making this antimicrobial mechanism a promising high-efficacy, low-cytotoxic option for treating bacterial and fungal infections.

To this end, biodegradable nanomaterials offer the advantage of breaking down into non-toxic byproducts, thereby reducing potential environmental impacts⁹⁷ and toxicity concerns.^98–100 With the inadvertent rise in microbial resistance, it's become crucial to identify newer, safer, potent treatment regimens in the field of medicine. Recently, few studies have shown that transition-metal based nanoparticles have antipathogenic properties.¹⁰¹ Iron-based nanomaterials, such as iron oxide nanoparticles – FeO or Fe₃O₄ – have shown promise in chemodynamic therapy treatment arena (CDT),^38,102–104 which facilitates intracellular Fenton-like reactions. In these reactions, iron ions (Fe²⁺) catalyse the decomposition of hydrogen peroxide (H₂O₂),^105,106 naturally produced within cells, leading to the formation of hydroxyl radicals (˙OH), a critical component of ROS. Additionally, one study has explored its anti-inflammatory and anti-biofilm properties^107,108 on a single Gram-positive strain of bacteria, whilst subjecting the flakes to hydrogen peroxide to facilitate the forced release of ROS through Fenton-like reactions. The focus of this particular study was to understand if pH changes in the surrounding tissue conditions would lead to an accelerated release of ROS, and its subsequent antimicrobial and anti-inflammatory property. The ambient degradation of the flake under atmospheric conditions was not included as such.

In this work, we demonstrate that FePS₃ nanoflakes exhibit significant antimicrobial efficacy against both Gram-negative and Gram-positive drug-resistant bacteria. Specifically, we observed near-complete inhibition of bacterial growth in both MRSA and drug-resistant variant of P. aeruginosa at extremely high bacterial concentrations—an OD₆₀₀ of 0.5, equivalent to 1 × 10⁸ cells per mL. In contrast, typical bacterial loads in natural infections range from 1 × 10² to 1 × 10⁷ cells per mL. This indicates that our experimental conditions rigorously challenge the antimicrobial efficacy of FePS₃, showcasing its potential as a robust antimicrobial agent. The material also demonstrated moderate inhibition against the fungal pathogen Candida albicans, suggesting differential susceptibility likely due to the complexity of fungal cell walls and the presence of ergosterol in their membranes. The antifouling mechanism exhibited by the FePS₃ flakes also helps in reducing the bacterial burden at the sites of infections. This dual action is particularly noteworthy as it highlights the compound's ability to address two critical aspects of bacterial contamination: not only eliminating the bacteria but also inhibiting their ability to adhere and form biofilms. The prevention of biofilm formation is crucial, as biofilms can lead to persistent infections and are often resistant to conventional antimicrobial treatments.

Reactive oxygen species (ROS) are natural byproducts of cellular metabolism. They play an important role in cellular homeostasis and signalling.¹⁰⁹ It includes radicals such as singlet oxygen (¹O₂), superoxide (O₂⁻), and hydroxyl (OH˙), which can cause oxidative stress in microbial cells and damage proteins, lipids, and DNA, leading to cell death. The primary mechanism by which FePS₃ generates ROS appears to be through the breakdown of the material itself, which disrupts cell membranes and causes cellular damage. While interactions with hydrogen peroxide, potentially through Fenton-like reactions, may contribute to ROS production, this is likely a secondary effect. Importantly, the material selectively targets microbial cells without harming mammalian cells, suggesting a specific mechanism that spares host tissues. Our work focusses on its degradative property under atmospheric conditions, leading to the formation of radicals. The exact mechanism is still unknown, but it could be possibly due to a combination of hydrolytic,¹¹⁰ oxidative,¹¹¹ and Fenton-like reaction. Mesoporous structures have been shown to enhance the photocatalytic activity of transition metal oxides, such as α-Fe₂O₃, due to their high specific surface area and redox activity.¹¹² Additionally, acidic environments have been known to cause an increase in ROS productions. This is presumably due to an increase in the leaching of metal ions when they come in contact with nanoparticles. Specific ROS generation tests were conducted to elucidate the mechanism underlying the antimicrobial activity of FePS₃. The nanoflakes exhibited significant ROS generation upon exposure to atmospheric conditions for two hours, with peak shifts associated with O₂˙⁻ and OH˙ seen. The presence of ROS causes oxidative stress in microbial cells, leading to cell membrane damage and eventual cell death. This mechanism is likely responsible for the observed antimicrobial activity and provides a plausible explanation for the efficacy of FePS₃ against various microbial strains. The ability to generate ROS makes FePS₃ a potent antimicrobial agent, leveraging oxidative stress to achieve microbial inhibition. The ROS generated by 2D-TMPX nanoflakes originates at the interface between the nanoflakes, solution, and microbial cells, which is then subsequently released into the surrounding medium. This is supported by the nanomaterial characterisation, which shows material breakdown as a function of time, as well as spectroscopic analysis of the ROS sensitive dyes – methylene blue, xanthine oxidase, and horse radish peroxidase. There is likely a concentration gradient, from surface coated FePS₃ to bulk solution, meaning that bacteria and fungi in direct contact with the nanomaterial experience higher levels of ROS exposure; however, deconvoluting the exact ROS gradient is beyond the scope of this work. Additionally, ROS generation is not observed as a microbe-released by-product, and it remains consistent across different microbial species, as interspecies variations are not present under these conditions. This nanomaterial, interface-bound ROS production ensures targeted antimicrobial efficacy without species-dependent fluctuations. The biocompatibility of the nanoflake also plays a crucial role in its further optimisation. HaCaT cells showed no cell death when treated with FePS₃ for 24 h and 48 h, with more than 95% viability observed in the cell counts. This is in line with other studies that show the viability of the compound against different cell types. Furthermore, it's seen that iron-based nanoparticles tend to cause ferroptosis in the bacteria, or selective accumulation of Fe-dependant in the cell, leading to an increase cell necrosis. Interestingly, these cells only exhibit selective cytotoxicity, with a greater impact on cancer cell line than normal fibroblast cells in the system.¹¹³

The molecular dynamics simulations also offer a deeper understanding of the stability and degradation patterns of the material under different environmental conditions, reinforcing the experimental observations of biodegradability and antimicrobial efficacy. They clearly demonstrate that pristine FePS₃ remains inert in the presence of O₂, whereas the introduction of a single sulphur defect catalyses a spontaneous reaction with O₂. This emphasizes the significant influence of structural imperfections on the chemical reactivity of the compound. The spontaneous reaction observed at the defect site suggests that defect engineering could be an important strategy to enhance the catalytic properties of FePS₃. By intentionally introducing defects, it may be possible to tailor the material's reactivity for specific applications, such as in catalysis or antimicrobial treatments. The defects could act as catalysts in the presence of oxygen, leading to increased ROS production, which in turn could more effectively kill bacteria and other pathogens. Alternatively, it would be possible to make FePS₃-based antimicrobial agents which could get activated under acidic conditions. Our study demonstrates that FePS₃ nanoflakes exhibit potent antimicrobial activity against drug-resistant bacteria, with moderate efficacy against fungi, largely driven by ROS generation. The biocompatibility and selective cytotoxicity further underscore its potential as a safe and effective antimicrobial agent. Additionally, the findings suggest that structural defect engineering could enhance its reactivity, opening avenues for targeted applications in both antimicrobial and catalytic fields. These results position FePS₃ as a promising candidate for developing advanced antimicrobial strategies.

4. Conclusion

This study shows the significant antimicrobial potential of FePS₃ nanoflakes against various multidrug-resistant pathogens. The material can be mechanically exfoliated and easily coated onto medically relevant instruments. Initial antimicrobial tests demonstrated that nanoflakes effectively inhibit the growth of bacterial (MRSA and P. aeruginosa) and fungal strain (C. albicans). CLSM images confirmed these results, showing increased cell death at multiple concentrations of the microbes, with the compound exhibiting an antifouling property. The degradation of the compound also takes place at a rapid pace, with 2 h of exposure to atmospheric conditions producing ROS species, which majorly contributes to its antimicrobial potency. Moreover, the degradative nature of FePS₃ could enhance its bioavailability and facilitate quicker clearance from the body. Further optimisation of the material will be performed to expand its antimicrobial potency to a broader spectrum of bacteria and fungi. The lowered antimicrobial activity on the fungal strains would require us to improve the drug delivery of the nanoparticle, which could help the material to pass through the thicker cell wall of the fungi. In conclusion, FePS₃ can be regarded as a next-generation antimicrobial agent which could be further optimised to develop a safer, more potent nanomaterial-based antimicrobial systems.

5. Materials and methods

5.1 Synthesis of FePS₃ nanoflakes

The FePS₃ crystals were purchased from Ossila Chemicals. Mechanical exfoliation of the bulk crystals was performed using the Scotch tape method.^114,115 Flake size consistency was confirmed by conducting repeated exfoliations under identical conditions, and flake sizes were characterized using SEM, AFM and TEM. Our findings indicate minimal variation between batches, with an average lateral size of approximately 2–4 μm and standard deviations within acceptable limits. This ensures that our findings are reproducible and reliable across different synthesis batches. The flakes were exfoliated onto 24-well plates (Thermo Fisher Scientific), and microbial testing was performed. For the SEM preparation, the samples were mechanically pressed onto a plasma-etched SiO₂/Si wafer as a substrate and examined.⁴⁸

5.2 Bacterial and fungal culture and sample preparation

The bacterial (MRSA & P. aeruginosa) and fungal strain (C. albicans) were obtained from the American Type Culture Collection. The strains and their ATCC numbers examined in this study were P. aeruginosa ATCC 27853, MRSA ATCC 700699, and C. albicans 16-24136919. These species were selected because of their medical relevance as pathogenic species and because they include representatives from both Gram-negative and Gram-positive bacteria. This ensured coverage of the two main bacterial taxonomies. For each experimental procedure, the overnight incubation of the bacterial cells in LB media were conducted at 37 °C. They were then inoculated using a loop and resuspended in LB broth and incubated overnight at 37 °C. The density of the bacterial suspensions in LB broth was adjusted to an OD₆₀₀ of 0.1 ± 0.04 (approximately 2 × 10⁷ cells per mL) during the logarithmic growth phase. For the concentration-dependent killing kinetics assay, the density of bacteria suspended in LB agar was adjusted to an O.D₆₀₀ of 0.05, 01 and 0.5. Tetracycline (TET) and fluconazole (FLUC) were obtained from Sigma-Aldrich and prepared as stock solutions at concentrations of 3 mg mL⁻¹ for tetracycline and 2 mg mL⁻¹ for fluconazole. These stocks were subsequently diluted to a series of concentrations, beginning at 128 μg mL⁻¹ and followed by 64 μg mL⁻¹, 6.4 μg mL⁻¹, and 0.64 μg mL⁻¹, corresponding to the minimum inhibitory concentration (MIC) values relevant for the bacterial and fungal strains under study. For each assay, equal volumes (50 : 50) of the bacteria (0.1 O.D₆₀₀) and the diluted antibiotic solution were combined and incubated at 37 °C for 24 hours. After incubation, serial dilutions were performed on the samples, and the bacterial or fungal colonies were enumerated to assess antimicrobial activity.

5.3 Characterization

Scanning electron micrographs were acquired using a field-emission scanning electron microscope. Specifically, imaging was conducted using an FEI Verios scanning electron microscope (VP, Oberkochen, BW, Germany) at an accelerating voltage of 5 kV following established methodologies.¹¹⁶

Transmission electron microscopy (TEM) images were captured using a JEOL 2100F microscope (JEOL, Musashino, Akishima, Tokyo, Japan). The microscope was operated at an acceleration voltage of 100 keV. Image processing and analysis were performed using Digital Micrograph 2.31 software.

Electron dispersive X-ray spectroscopy was performed using an Oxford X-Maxⁿ attachment on a JEOL 2100D microscope (JOL; Musashino, Akishima, Tokyo, Japan). Analysis and processing of the images were performed using the Aztec software.

ATR-FTIR was performed using a Bruker Alpha II Spectrometer. The spectra ranged from 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹ and 32 average scans.

Atomic force microscopy images were taken and processed using a Cypher ES atomic force microscope (Oxford Instruments, Asylum Research, Santa Barbara, CA, USA). Imaging was conducted under ambient conditions. For all measurements, AC240 cantilevers (Oxford Instrument, Asylum Research, Santa Barbara, CA, USA, nominal spring constant k_c = 2 N m⁻¹) were utilized. When operated in the AC mode, imaging forces were minimized by maintaining a setpoint ratio [imaging amplitude (A)/free amplitude (A₀)] greater than 0.7.

5.4 CLSM imaging

Confocal laser scanning microscopy (CLSM) was performed to determine the number and state of bacterial and fungal cells using a fluoview FV1200 inverted microscope, Olympus, Tokyo, Japan, and ZEISS LSM 880 Airyscan upright microscope, Oberkochen, Germany. The flakes were mechanically exfoliated and pressed onto a 24-well plate (Thermo Fisher Scientific), and the bacterial and fungal cells were inoculated into it and incubated overnight. The cells were then stained using a LIVE/DEAD BacLight bacterial viability kit, which included SYTO 9 and propidium iodide (Molecular Probes, Invitrogen, Grand Island, NY, USA). SYTO 9 binds to nucleic acids in intact and damaged cell membranes and fluoresces green. PI specifically binds to damaged cells and fluoresces red, thus differentiating them from the remaining cells. Photobleaching was prevented using single-surface confocal scanning. The ratio of live cells to dead cells was quantified using Cell C software.

5.5 Hemolysis

Hemolysis was performed to determine the percentage hemolytic activity elicited by the compound. 1 mL of patient blood sample collected in vacutainer tubes was diluted to 10% using PBS and stored at 37 °C. 200 μL of diluted blood was added to a 24-well plate (Thermo Fisher Scientific) containing mechanically exfoliated FePS₃ flakes and incubated overnight at 37 °C. The samples were then centrifuged to separate the supernatant, which was carefully collected for analysis. The absorbance of the supernatant was measured at 600 nm using a UV-visible spectrophotometer (CLARIOSTAR) to determine the extent of hemolysis induced by the FePS₃ flakes. The absorbance values obtained were compared to those of an untreated control sample, which contained the diluted blood without any FePS₃ flakes. This comparison allowed us to assess the relative hemolytic activity of the compound. The percent hemolysis was calculated and subtracted from 100% representing the percentage of cells that remained intact (live cells).

5.6 Cytotoxicity

The cytotoxicity of FePS₃ was assessed by incubating the material with human immortalized keratinocyte (HaCaT) cells for 24 and 48 h and assessing cell viability compared to an untreated control. Cell viability was determined using the resazurin metabolic assay, which relies on the ability of viable cells to convert the non-fluorescent resazurin dye into the fluorescent derivative resorufin. Mechanically exfoliated FePS₃ was pressed onto the base of triplicate wells of a 24-well plate using tape. After UV sterilization of the prepared plate, 500 μL of HaCaT cells at a concentration of ∼2 × 10⁵ cells per mL was then added to triplicate wells either with or without FePS₃ and incubated for 24 h at 37 °C. This was repeated with a 48-hour incubation period, but with half the cell concentration of ∼1 × 10⁵ cells per mL to ensure that the cells did not become excessively confluent. The culture medium used was Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 1% penicillin/streptomycin (Gibco) and 10% foetal bovine serum (FBS). After incubation, the medium was removed, and the cells were incubated with resazurin at a concentration of 0.015 mg g⁻¹ for 1 h. Using a microplate reader (CLARIOSTAR), the fluorescence of the samples was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The comparison was done between the treated and untreated cells.

5.7 Detection of ROS

The production of oxidative species through the breakdown of few-layer FePS₃ was assessed using various dyes, using similar protocols from a prior study.⁶⁰ Specifically, xanthine oxidase (XO) was employed to detect superoxide radicals, HRP, or horse radish peroxidase was used to detect the hydroxide radicals, and singlet oxygen (¹O₂) was detected using methylene blue (MB). The few-layer FePS₃ was exfoliated onto the base of a 24-well plate prior to testing. 5 mg HRP was added to 50 mL of Milli-Q water solution to make up HRP solution, 5 μM oxidase was mixed with in 50 mL of Milli-Q water to prepare xanthine oxidase solution, and 100 mg of methylene blue was added into 50 mL of Milli-Q water to make up the MB solution., following which 2 mL of all three solutions were added and incubated for 2 h under atmospheric conditions. Post incubation, analysis was performed using a UV spectrophotometer (CARY 3500) across 200–800 nm wavelength. The absorbance peaks for ¹O₂, OH⁻, and O₂˙⁻ were observed at 550–700 nm, 350–450 nm, and 250–350 nm respectively.

5.8 Computational methods

Computational analyses were conducted utilizing the Vienna ab initio simulation package (VASP 5.4.4).¹¹⁷ The generalized gradient approximation (GGA) framework was applied, incorporating the Perdew–Burke–Ernzerhof (PBE) exchange–correlation functional¹¹⁸ and projector augmented wave (PAW) methodology¹¹⁹ to define the ion–electron interaction. An energy cutoff of 466 eV was utilized, with a k-point mesh configuration of 5 × 5 × 1 for geometry optimization, and solely the gamma point for ab initio molecular dynamics (AIMD) to sample. The FePS₃ unit cell, characterized by lattice parameters a = 5.947 Å, b = 10.3 Å, and c = 6.722 Å, was sourced from the work of Ouvrard et al.¹²⁰ A defect layer was created by removing a single sulphur atom from the surface. A 15 Å vacuum spacer was introduced along the z-axis to mitigate interlayer interactions. For geometry optimization, atomic positions were relaxed until the system's total energy converged to 10⁻⁴ eV, and the Hellmann–Feynman forces on each atom were reduced to less than 0.01 eV Å⁻¹. In AIMD simulations, two water molecules and two O₂ molecules were included in each system, with all atoms allowed to relax throughout the simulation. These simulations were performed at 300 K with a timestep of 0.5 fs.

5.9 Statistical analysis

Data visualization and statistical analyses were conducted using paired t-tests and one-way ANOVA in GraphPad Prism 10.0.0 for Windows (GraphPad Software, Boston, Massachusetts, USA, https://www.graphpad.com). Each test was performed with a minimum of three independent replicates. The statistical tests applied, along with the corresponding P values, are specified in the figure legends and within the figures.

Data availability

All relevant data are within the manuscript and its additional files. Further clarification on data is available upon request from the authors.

Conflicts of interest

The authors declare no conflicts of interest related to this research.

Acknowledgements

The authors would like to express their sincere gratitude to the RMIT Microscopy and Microanalysis Facility for their assistance with scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). We also extend our thanks to the Micro Nano Research Facility at RMIT University for providing access to atomic force microscopy (AFM). This research was supported by the Australian Government through the Discovery Early Career Researcher Award (DECRA – DE220100511) and (DP220100020) scheme and the Research Training Programme (RTP) Scheme. We are also grateful for the computational resources provided by the National Computational Infrastructure (NCI) and the Pawsey Supercomputing Research Centre, through the National Computational Merit Allocation Scheme (project kl59 and resource grant uo96).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nr03409k

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