Abstract Ti₃C₂–Co₃(PO₄)₂ nanocomposite was synthesized by a microwave-assisted method and evaluated for its physicochemical characteristics, antifungal, antioxidant and anti-inflammatory responses, cell viability and short-term stability under simulated salivary conditions. XRD, FTIR, XPS, TEM/HRTEM, SAED and UV–Vis DRS analyses supported the formation and physicochemical characteristics of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite. Agar well diffusion and broth microdilution assays demonstrated antifungal activity against Candida albicans and Aspergillus flavus. At 1,000 µg/well, inhibition zones of 20 and 17 mm were obtained against C. albicans and A. flavus, respectively, compared with 23 and 21 mm for fluconazole. The corresponding MIC/MFC values were 62.5/125 µg/mL and 125/250 µg/mL. DPPH radical-scavenging activity increased from 14.33 ± 4.04% at 5 µg/mL to 90.00 ± 2.00% at 100 µg/mL, compared with 94 ± 2% for ascorbic acid. In LPS-stimulated RAW 264.7 macrophages, NO inhibition reached 82 ± 3%, while TNF-α, IL-6, IL-1β and intracellular ROS levels decreased to approximately 22–30% of the control values. Cell viability remained above 80% in RAW 264.7 macrophages across the tested concentration range. After 48 h exposure to simulated saliva, weight retention ranged from 95 ± 1% to 100 ± 1% across pH 5.5–7.5, indicating short-term stability based on weight retention. Overall, the findings provide preliminary evidence of multifunctional biological activity and short-term physicochemical stability of Ti₃C₂–Co₃(PO₄)₂ under the tested conditions, supporting further investigation of the material for oral biomaterial applications.

Keywords: Ti₃C₂ MXene, Cobalt phosphate, Nanocomposite, Antifungal activity, Antioxidant activity, Anti-inflammatory response, Salivary stability

Citation:  Majety D.G., Anbalagan, G., Kannan, I.I.G., Suresh, V., and Subramanian, B. 2026. Ti₃C₂–Co₃(PO₄)₂ nanocomposite with antifungal, antioxidant and anti-inflammatory activities and short-term stability under simulated salivary conditions. Natural and Life Sciences Communications. 25(4): e2026100.

Graphical Abstract:

INTRODUCTION

Oral and craniofacial tissues are continuously exposed to diverse microbial communities, and the emergence of antimicrobial resistance poses a critical challenge to oral healthcare. Fungal infections such as oral candidiasis and denture stomatitis have become increasingly prevalent, particularly among immunocompromised individuals including cancer patients, transplant recipients, and the elderly (Kalaivani et al., 2025). Oral fungal infections, particularly candidiasis, represent an important clinical concern, especially among immunocompromised individuals. Increasing antifungal resistance further complicates their management and highlights the need for alternative materials with antifungal activity (Lee et al., 2021). Oral tissues are continuously exposed to diverse microbial communities, and fungal infections remain an important concern in susceptible individuals. Candida albicans is a major opportunistic oral pathogen associated with candidiasis and denture-related infections, whereas Aspergillus flavus can cause opportunistic infection in immunocompromised individuals. Fungal colonization of dentures and oral surfaces may contribute to persistent inflammation and complicate treatment. Increasing resistance to conventional antifungal agents has encouraged the development of alternative materials with complementary antimicrobial and protective functions (Desai et al., 2026).

Candida albicans, normally a commensal organism, becomes pathogenic under immune suppression, while Aspergillus flavus is an opportunistic fungal species that can cause clinically important infections, particularly in susceptible individuals. Fungal biofilm formation on dentures, implants, and mucosal surfaces aggravates inflammation and reduces antifungal efficacy (Kashyap et al., 2024). Conventional drugs such as azoles and polyenes often exhibit drawbacks including resistance, cytotoxicity, and poor penetration into fungal biofilms. Hence, the development of multifunctional biomaterials capable of inhibiting fungi, neutralizing oxidative stress, and modulating inflammation represents a major advancement in restorative and regenerative dentistry (Amann et al., 2025). Aspergillus flavus is an opportunistic fungal species that can cause clinically important infections, particularly in susceptible individuals. Its ability to persist under challenging environmental conditions and form interactions with biological surfaces highlights the importance of developing effective antifungal materials. Therefore, multifunctional biomaterials combining antifungal activity with antioxidant and anti-inflammatory properties may provide additional advantages for future oral healthcare applications.

Two-dimensional (2D) materials, especially MXenes, have recently gained attention for their distinctive physicochemical and biomedical properties. MXenes are a family of layered transition metal carbides, nitrides, or carbonitrides with the general formula M_n+1X_nT_x where M is an early transition metal, X is carbon and/or nitrogen, and Tₓ represents surface terminations such as –O, –OH, or –F. They are produced by selective etching of the A-element from precursor MAX phases, resulting in high surface area nanosheets with abundant functional groups (Devanshi et al., 2024). The antimicrobial activity of Ti₃C₂-based systems is mainly attributed to the direct disruption of microbial cell membranes, due to the ultrathin two-dimensional nanosheets and their sharp edges, which cause physical damage to the cell membranes and enhance their permeability (Tamhane et al., 2024).

Metal phosphates, particularly cobalt phosphate (Co₃(PO₄)₂), are attractive hybrid materials due to their stability, ionic tunability, and biological compatibility. Cobalt, an essential trace element and cofactor for vitamin B₁₂, contributes redox activity and catalytic potential to these materials. Co₃(PO₄)₂ demonstrates strong antimicrobial properties by disrupting microbial membranes and enzymes while inducing oxidative stress, yet remains cytocompatible when integrated within bioceramic matrices. Incorporating cobalt phosphate into titanium carbide MXene may combine the conductive, ROS-modulating properties of Ti₃C₂ with the redox and ion-releasing functions of Co₃(PO₄)₂, enhancing antifungal, antioxidant, and anti-inflammatory performance (Anwar et al., 2019; Harini et al., 2024; Thongjan et al., 2025). Ti₃C₂ MXene possesses a layered morphology, high accessible surface area and surface terminations that can facilitate interactions with microbial cells. In Ti₃C₂-based systems, direct contact with microbial membranes and surface-associated oxidative effects have been proposed as possible contributors to antimicrobial activity. Co₃(PO₄)₂ provides a complementary inorganic phase with redox-active cobalt centres and phosphate groups. In the present composite, these features may contribute to the observed biological responses; however, the relative contributions of membrane disruption, ROS generation, electron transfer and cobalt-ion release were not directly investigated.

In the present Ti₃C₂–Co₃(PO₄)₂ nanocomposite, the antifungal response may arise from complementary effects associated with the Ti₃C₂ nanosheet morphology, surface functional groups, and cobalt phosphate phase. Direct contact between the nanocomposite and fungal cells may contribute to membrane-associated effects, while the chemical characteristics of the cobalt phosphate phase may additionally influence oxidative and ionic interactions. However, specific contributions from ROS generation, membrane disruption, electron transfer, or cobalt-ion release were not directly investigated in the present study. Although several MXene-based nanocomposites have been investigated for antimicrobial and biomedical applications, the present study specifically examines a Ti₃C₂–Co₃(PO₄)₂ composite from an oral-biomaterial perspective. The study integrates structural characterization with antifungal screening against C. albicans and A. flavus, DPPH radical-scavenging activity, inflammatory-response assays in LPS-stimulated RAW 264.7 macrophages, cell-viability assessment and short-term stability under simulated salivary conditions. The objective was to establish the physicochemical characteristics and preliminary multifunctional biological responses of Ti₃C₂–Co₃(PO₄)₂ under conditions relevant to oral biomaterial research. This integrated oral-biomaterial-oriented evaluation distinguishes the present system from previous MXene-based studies that primarily focused on energy storage, photocatalysis, or general antimicrobial screening (Rajasri et al., 2022; Hadia et al., 2025). The structure, morphology, and crystallinity were analyzed using X-ray diffraction (XRD), transmission electron microscopy (TEM), and high-resolution TEM (HRTEM). The biological efficacy of the composite was assessed through antifungal assays against Candida albicans and Aspergillus flavus, antioxidant evaluation by DPPH radical scavenging, and anti- inflammatory studies in LPS-stimulated RAW 264.7 macrophages. Furthermore, pH- dependent stability under simulated salivary conditions (pH 5.5–7.5) was evaluated to determine suitability for intraoral environments. In this study, a microwave-assisted Ti₃C₂–Co₃(PO₄)₂ nanocomposite is introduced, which is specifically synthesized for oral biomedical applications. It is a multifunctional platform that combines antifungal, antioxidant, anti-inflammatory, cell viability and saliva-pH stability evaluations uniquely. This combination of properties provides a basis for investigating Ti₃C₂–Co₃(PO₄)₂ as a multifunctional material for oral biomaterial research.

MATERIALS AND METHODS

Materials

Analytical-grade reagents were used without further purification: titanium aluminum carbide (Ti₃AlC₂), hydrochloric acid (HCl), lithium fluoride (LiF), cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O), disodium hydrogen phosphate (Na₂HPO₄), ethanol, acetone, and distilled water. All chemicals were procured from certified suppliers.

Synthesis of titanium carbide (Ti₃C₂)

Titanium carbide (Ti₃C₂) was prepared from the Ti₃AlC₂ MAX phase via selective etching of aluminum layers. LiF (3.0 g) was dissolved in 40 mL of 9 M HCl under stirring for 30 min to ensure complete dissolution. The LiF/HCl etching process was carried out in a fume hood with appropriate acid-resistant gloves, face protection, and controlled stirring to minimize exposure to acidic vapors. The synthesis was not performed under a fully inert atmosphere; however, oxidation was minimized by conducting the process at a controlled temperature, using freshly prepared etchant, repeated washing to neutral pH, ethanol rinsing, and immediate drying at 80°C. Ti₃AlC₂ (1 g) was then added gradually and maintained at 40°C for 48 h with continuous stirring. The product was repeatedly washed with distilled water until a neutral pH (~7) was obtained, followed by ethanol rinsing. The final precipitate was oven- dried at 80°C for 24 h to obtain Ti₃C₂ powder.

Synthesis of cobalt phosphate precursor (Co₃(PO₄)₂)

Cobalt (II) nitrate hexahydrate (2.91 g) was dissolved in 50 mL of distilled water to prepare Solution A. Separately, disodium hydrogen phosphate (2.13 g) was dissolved in 50 mL of distilled water to prepare Solution B. Solution B was added dropwise to Solution A under continuous stirring for 1 h, resulting in the formation of a light-purple cobalt phosphate precursor suspension, hereafter referred to as Solution C.

Preparation of titanium carbide–cobalt phosphate nanocomposite (Ti₃C₂–Co₃(PO₄)₂)

Ti₃C₂ (1 g) was first dispersed in 25 mL of distilled water and sonicated for 20 min to obtain a homogeneous Ti₃C₂ dispersion. The freshly prepared Ti₃C₂ dispersion was then added to Solution C containing the cobalt phosphate precursor under continuous stirring. The resulting Ti₃C₂–cobalt phosphate suspension was stirred for 3 h to ensure uniform mixing and effective interaction between Ti₃C₂ and the cobalt phosphate precursor. Subsequently, the reaction mixture was subjected to microwave irradiation using a laboratory microwave reactor operating at 2.45 GHz and a fixed power of 700 W for 10 min. The reaction was conducted in a microwave-compatible borosilicate reaction vessel with intermittent monitoring to minimize overheating and solvent loss. After microwave irradiation, the resulting precipitate was collected and thoroughly washed several times with distilled water, followed by ethanol and acetone, to remove unreacted precursors and residual impurities. The washed product was dried at 80°C for 24 h and subsequently calcined at 300°C for 3 h to obtain the Ti₃C₂–Co₃(PO₄)₂ nanocomposite.

Antifungal activity

The antifungal potential of Ti₃C₂–Co₃(PO₄)₂ was evaluated against Candida albicans and Aspergillus flavus using the agar well diffusion method. A stock solution (10 mg/mL) was prepared in dimethyl sulfoxide (DMSO). Fungal strains were cultured in Sabouraud Dextrose Broth (SDB) at 37°C for 2–3 h and spread onto Sabouraud Dextrose Agar (SDA) plates to establish lawn cultures. The 10 mg/mL stock suspension was loaded into the wells at 50, 75 and 100 µL, corresponding to 500, 750 and 1,000 µg/well, respectively. Fluconazole discs served as positive controls, while DMSO acted as the negative control. After incubation at 37°C for 24 h, inhibition zones were measured (mm) to determine antifungal efficacy.

Antioxidant activity

The antioxidant capability of the composite was assessed via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. A freshly prepared 0.1 mM ethanolic DPPH solution was mixed with varying concentrations (5–100 µg/mL) of Ti₃C₂–Co₃(PO₄)₂ suspended in phosphate-buffered saline (PBS, pH 7.4). The mixtures (1 mL each) were incubated for 30 min in the dark at room temperature, and absorbance was recorded at 517 nm using a UV–Vis spectrophotometer. To correct for optical interference from the nanocomposite suspension, concentration-matched sample blanks containing Ti₃C₂–Co₃(PO₄)₂ in PBS without DPPH were measured at 517 nm. The corresponding blank absorbance was subtracted from the absorbance of the sample-containing DPPH reaction before calculating radical-scavenging activity. Ascorbic acid served as the reference standard. Radical scavenging efficiency was calculated using the formula: DPPH scavenging activity (%) = [(A_control − A_sample) / A_control] × 100. All measurements were performed in triplicate, and data were expressed as mean ± SD.

Anti-inflammatory activity

Anti-inflammatory effects were examined using the RAW 264.7 murine macrophage cell line (ATCC TIB-71) stimulated with lipopolysaccharide (LPS). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C in a 5% CO₂ atmosphere. Cells (1 × 10⁵ cells/well) were seeded into 96-well plates and pre-treated with Ti₃C₂–Co₃(PO₄)₂ (5–100 µg/mL) for 2 h before stimulation with LPS (1 µg/mL, E. coli O111:B4) for 24 h. Dexamethasone (10 µM) was used as a positive control, and untreated cells served as negative controls. Cell viability was determined using the MTT assay. After incubation with MTT (5 mg/mL, 4 h), formazan crystals were dissolved in DMSO, and absorbance was recorded at 570 nm. Nitric oxide (NO) levels were measured using the Griess reaction by mixing culture supernatant with equal volumes of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride), followed by absorbance reading at 540 nm. Pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) were quantified using commercial ELISA kits. Intracellular reactive oxygen species (ROS) were analyzed with the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe (10 µM, 30 min incubation). Fluorescence was measured at excitation/emission wavelengths of 485/535 nm. The MTT measurements were available as mean ± SD values. The NO, cytokine and intracellular ROS results were available as summarized values at each concentration and were therefore presented descriptively.

pH stability and saliva simulation test

The oral environmental stability of Ti₃C₂–Co₃(PO₄)₂ was evaluated using simulated saliva prepared according to the Fusayama–Meyer formulation (0.4 g/L NaCl, 0.4 g/L KCl, 0.795 g/L CaCl₂·2H₂O, 0.78 g/L NaH₂PO₄·2H₂O, 0.005 g/L Na₂S·9H₂O, and 1 g/L urea). The pH was adjusted to 5.5, 6.5, and 7.5. Nanocomposite samples (10 mg) were suspended in 20 mL of simulated saliva and incubated at 37°C for 48 h to simulate short-term oral exposure. Samples were then centrifuged, dried, and analyzed for visual changes, pH variation, and weight retention. The stability index (%) was determined as: Stability index (%) = (W₁/W₀) × 100, where: W₀ = initial dry weight of the sample, W₁ = final dry weight after saliva exposure. UV–Vis absorbance at 280 nm was recorded to monitor potential leaching of metal or phosphate ions.

Statistical analysis

Data are presented as mean ± standard deviation (SD) where replicate-level measurements were available. The DPPH dataset contained three measurements per concentration and was therefore presented as mean ± SD. The MTT and pH-stability datasets were analyzed using the statistical procedures described above where replicate-level data were available. The NO, cytokine and intracellular ROS datasets were available as single summarized values at each concentration; therefore, these results were presented descriptively and inferential statistical analysis was not performed for these datasets.

RESULTS

Materials characterization

The physicochemical characteristics of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite were systematically examined by XRD, FTIR, UV–Vis spectroscopy, and electron microscopy (Figure 1). FTIR spectra were recorded using a Shimadzu IRTracer-100 FTIR spectrophotometer in the range of 4,000–400 cm⁻¹, with a spectral resolution of 4 cm⁻¹ and 32 scans per sample. Background correction was performed before each measurement.

X-ray diffraction (XRD) analysis

The X-ray diffraction (XRD) pattern of Ti₃C₂–Co₃(PO₄)₂ nanocomposite, demonstrates that the cobalt phosphate has been successfully incorporated in the Ti₃C₂ MXene matrix and the characteristic layer structure of the Ti₃C₂ parent MXene is retained in the Ti₃C₂–Co₃(PO₄)₂ nanocomposite (Figure 1). The diffraction peak at about 2θ = 9–10°, assigned to the (004) plane, along with the reflection at 25–26°, assigned to the (006) plane, are typical feature of Ti₃C₂ MXene, indicating an enlarged interlayer spacing as a consequence of selective etching and surface functionalization. These reflections are in good agreement with the diffraction pattern of Ti₃C₂ MXene reported (PDF/JCPDS No. 52-0875), which shows that the two dimensional layer structure is unchanged after the composite is formed. The large background intensity and relatively low diffraction intensity suggest that the Ti₃C₂ sheets have a partially exfoliated structure and lower long-range crystallinity, which is a common characteristic of few-layer MXenes. In addition to the Ti₃C₂ reflections, several distinct diffraction peaks located at approximately 18.5°, 20.8°, 22.6°, 29.3°, 33.5°, 35.8°, 41.7°, 45.4°, 49.8°, 53.1°, 57.8°, 60.2°, 64.3°, and 73.2° are indexed to the (020), (200), (130), (131), (141), (301), (341), (350), (161), (132), (501), (181), (262), and (291) crystallographic planes, respectively, corresponding to crystalline cobalt phosphate (Co₃(PO₄)₂). The reflections are consistent with a well crystallised cobalt phosphate phase and no other reflections from secondary cobalt oxide or metallic cobalt impurities are detected and in good agreement with the JCPDS/PDF card No. 13-0503. The diffraction peaks of both MXene and cobalt phosphate peaks verify the heterostructure formation instead of the bulk phase. Moreover, moderate peak broadening observed for cobalt phosphate peaks indicates that nanosized crystallites are uniformly attached to the surface of Ti₃C₂. The intimate interfacial interaction between partially crystalline Ti₃C₂ structure and highly crystalline Co₃(PO₄)₂ nanoparticles is expected to promote the electron transfer rate, enhance the density of electrochemically active sites and enhance the structural stability of the nanocomposite, making it a promising material for functional biomedical applications.

Figure 1. XRD pattern of Ti₃C₂–Co₃(PO₄)₂ showing indexed Ti₃C₂ and Co₃(PO₄)₂ peaks.

FTIR analysis

The Fourier Transform Infrared (FTIR) spectrum of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite is further analyzed to confirm the successful formation of Co₃(PO₄)₂ on the surface of Ti₃C₂ MXene, which is evident from the presence of characteristic vibrational bands associated with the surface functional groups and phosphate linkages (Figure 2). FTIR measurements were performed using samples obtained from three independently synthesized batches to assess reproducibility of the synthesis. The wide absorption band around 3,448.92 cm^-1 is assigned to the hydroxyl (–OH) stretching vibrations of the adsorbed water molecules, and surface hydroxyl functional groups, showing the hydrophilic characteristic of the etched MXene sheets and composites. The weak band at 3,052.86 cm⁻¹ is attributed to the C–H stretching vibrations, which can be attributed to the presence of residual organic species or carbon-containing surface terminations. The band at 1,579.16 cm⁻¹ may be associated with the bending vibration of adsorbed H–O–H and/or surface oxygen-containing species; therefore, a definitive C=O assignment is avoided. The strong absorption bands at 1,032.55, 973.74, 937.00 and 839.92 cm^-1 are identified as the asymmetric and symmetric stretching vibrations of P–O bonds in PO₄³⁻ tetrahedral units, which is typical for crystalline cobalt phosphate. The peak at 698.34 cm⁻¹ is assigned to the O–P–O bending vibration of phosphate groups, and the strong absorption band at 536.62 cm⁻¹ is assigned to the Co–O stretching vibration corresponding to the CoO₆ octahedral coordination, which indicates the presence of cobalt phosphate. The simultaneous detection of vibrations from the Ti₃C₂ surface functional groups and the characteristic vibrations for phosphate and Co–O confirms the anchoring of the Co₃(PO₄)₂ nanoparticles on the surface of the Ti₃C₂ without destroying the characteristic MXene framework. In addition, no other additional impurity-related absorption bands were observed, revealing that the composite synthesized is of high purity.

Figure 2: FTIR spectrum showing O–H, oxygen-containing surface, phosphate (PO₄³⁻), and Co–O-related vibrational bands.

XPS analysis

The X-ray photoelectron spectroscopy (XPS) analysis was performed to investigate the surface elemental composition and chemical states of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite, thereby confirming the successful integration of cobalt phosphate onto the Ti₃C₂ MXene framework (Figure 3a–f). The high chemical purity of the synthesized material is clearly reflected in the absence of impurity elements in the survey spectrum. A survey spectrum shows the presence of Ti, C, Co, P, and O without detectable impurities, indicating high chemical purity of the synthesized material. The strong Ti and C signals are from the Ti₃C₂ MXene, while the signals of Co, P, and O are from the successful deposition of cobalt phosphate onto the surface of the Ti₃C₂ MXene. The absence of any other peaks which might be due to unwanted metallics or oxides further suggests that the microwave-assisted synthesis provided a well-defined heterostructure. The characteristic doublets pertaining to Ti–C and surface Ti–O species are observed in the high-resolution Ti 2p spectrum. The higher energy peaks at ~458.7 eV (Ti 2p₃/₂) and ~464.6 eV (Ti 2p₁/₂) are assigned to oxidized Ti species that were created by surface termination, and the lower binding energy peaks at ~456.8 eV are assigned to Ti bonded in the Ti₃C₂ lattice. The presence of both Ti–C and Ti–O environments suggests that the MXene still has the layered carbide structure and has oxygen-containing functional groups to enhance the bonding of the interfaces with cobalt phosphate. The C 1s spectrum could be separated into several components at about 282.7, 284.6, 286.6 and 288.9 eV, representing C–Ti, graphitic C–C/C=C, C–O and O–C=O species, respectively. The high intensity of the carbon species C–Ti demonstrates the preservation of the Ti₃C₂ framework, while the presence of oxygen in the carbon species is due to surface functionalization and minor atmospheric oxidation, which are needed to anchor Co₃(PO₄)₂ nanoparticles. The Co 2p spectrum shows the characteristic spin–orbit doublet of cobalt with peaks centered at 781.0 eV (Co 2p₃/₂) and 803.6 eV (Co 2p₁/₂), as well as well-defined satellite peaks at 786.1 eV and 803.6 eV. These features are typical of Co²⁺ species, and it is known that there is only a small amount of cobalt in the 3+ oxidation state in the phosphate lattice. The lack of a metallic cobalt resonance further confirms that the cobalt has been chemically incorporated into the phosphate framework and not formed into elemental cobalt nanoparticles. Two fitted components are located around 133.6 eV and 132.1 eV, which are attributed to the phosphate P–O bonding and the interfacial P–O–Co bonding, respectively, and represent the P 2p spectrum. The binding energies are typical for PO₄³⁻ tetrahedral units, and indicate the presence of crystallized cobalt phosphate and strong chemical interaction between the cobalt ions and the phosphate groups. The O 1s spectrum is decomposed into three peaks at ~530.2, ~532.0 and ~533.6 eV, corresponding to the lattice O, Ti–O and surface hydroxyl or surface water, respectively. Simultaneous presence of these oxygen species indicate the presence of strong Ti–O–P interfacial linkages and a large number of oxygen containing functional groups which increase the hydrophilicity and interfacial stability. In general, the XPS results clearly demonstrate the formation of the Ti₃C₂–Co₃(PO₄)₂ heterostructure and show that the two components are well coupled electronically on the conductive MXene material support. This interfacial coupling is expected to enable efficient electron transport, provide greater redox activity, structural stability and play a significant role in the remarkable antifungal, antioxidant, anti-inflammatory and cell viability properties obtained for the synthesized nanocomposite. XPS analysis was performed to further evaluate the elemental composition of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite. The survey spectrum showed the characteristic Ti, C, Co, P and O signals, while no detectable Al signal was observed within the detection limit of the XPS measurement. Therefore, No Al signal was detected within the detection limit of XPS, supporting effective removal of Al during the etching process.

Figure 3. XPS spectra of Ti₃C₂–Co₃(PO₄)₂ nanocomposite: survey spectrum (a) and high-resolution XPS spectra of Ti 2p (b), C 1s (c), Co 2p (d), P 2p (e), and O 1s (f). 

UV–visible analysis

The Ti₃C₂–Co₃(PO₄)₂ nanocomposite exhibits a wide-range UV–visible diffuse reflectance spectroscopy (UV–Vis DRS) spectrum, indicating that it is effective in harnessing both the ultraviolet and visible light (Figure 4). A remarkably strong edge of absorption is seen at 200–280 nm, mostly due to the intrinsic electronic transitions of the Ti₃C₂ MXene framework and charge-transfer transitions through Ti–C bonds. After this, the absorption intensity goes down from 300 to 400 nm, which means that the composite has a response to the visible light spectrum. The presence of cobalt phosphate is indicated by the presence of a broad and distinct absorption band from around 450 to 680 nm and a maximum absorption band near 580–600 nm. The absorption in the visible region is due to d–d electronic transitions of Co²⁺ ions and ligand to metal charge transfer (LMCT) between the phosphate oxygen atoms and cobalt centers. The width of the absorption band suggests that Ti₃C₂ and Co₃(PO₄)₂ are well coupled electronically. The lack of sharp absorption edges also indicates the formation of a heterostructured nanocomposite with a high amount of surface states and defect sites. The metallic conductivity of Ti₃C₂ offers fast electron transport channels, and promotes charge-carrier migration across the interface. The broad absorption extending into the visible region indicates an extended optical response of the composite. However, UV–Vis DRS alone does not provide direct evidence of charge-carrier separation, interfacial electron transfer, or photocatalytic activity.

Figure 4. UV–Vis absorption with 200–280 nm and 450–680 nm.

TEM/HRTEM/SAED analysis

The transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) analyses reveal the successful formation of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite and establish the integrity of the structure at the nanoscale (Figure 5). The TEM image shows the thin, transparent and sheet-like Ti₃C₂ MXene layers with uniformly distributed cobalt phosphate nanoparticles. The Ti₃C₂ sheets still maintain the two dimensional sheet structure with some wrinkling and overlapping, suggesting that the layered structure has not been affected by the deposition of cobalt phosphate. The darker contrast regions are attributed to the higher electron density of Co₃(PO₄)₂ nanoparticles, while the lighter contrast is due to the ultrathin Ti₃C₂ nanosheets. The nanoparticles have anchoring ability on the surface of MXenes and less agglomeration, which can ensure intimate interfacial contact and have a beneficial effect on electron transmission speed. The Cobalt phosphate particles have a size range of 15-40 nm, while the Ti₃C₂ nanosheets are extended laterally with a range of several hundred nanometres, which creates an interconnected conductive network. The measured particle sizes ranged from approximately 15–40 nm, with an average size of 34.6 ± 6.8 nm based on more than 100 particles. The HRTEM image also confirms the crystalline nature of the composite due to the presence of distinct and continuous lattice fringes. The interplanar spacing of the (141) plane of crystalline Co₃(PO₄)₂ can be ~0.34 nm; for the layered Ti₃C₂ structure, the lattice fringes are parallel to each other, corresponding to the basal planes of the MXene sheets, with the interlayer spacing increased by functionalization at the surface. The sharp and distinct lattice fringes show high crystallinity of the deposited cobalt phosphate nanoparticles and the good interfacial coupling with the Ti₃C₂ support. This intimate contact is expected to enable efficient charge transfer across the heterointerface, while at the same time ensuring structural stability. The SAED pattern shows the presence of bright concentric diffraction rings consisting of many discrete diffraction spots and not continuous diffuse rings, which is a sign of polycrystalline structure of the nanocomposite. The diffraction rings correspond to the randomly oriented Co₃(PO₄)₂ nanocrystals and the layered Ti₃C₂ structure, which indicates that the phases are not mixed with the formation of undesirable impurity phases. The intensity and sharpness of the diffraction rings are high, showing good crystallinity, while the number of diffraction spots is large, suggesting the presence of nanosized crystallites with different orientations. The integrated TEM, HRTEM and SAED analysis therefore showed that the crystalline Co₃(PO₄)₂ nanoparticles were homogeneously immobilised onto the conductive Ti₃C₂ nanosheets and created a heterostructure with a large interface contact area, rich electrochemically active sites and an efficient electron migration pathway, which makes the composite material highly appropriate for Biological applications.

Figure 5. TEM (a), HRTEM (b), and SAED (c) images confirming granular morphology, lattice ordering, and polycrystallinity of the nanocomposite.

Antifungal activity

The inhibitory efficiency of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite was evaluated on the opportunistic fungal pathogens, Candida albicans and Aspergillus flavus by agar well diffusion method to assess its antifungal activity. The results shown in Figure 6A–B show that the nanocomposite produced measurable inhibition zones against both fungal strains, with the inhibition-zone diameter increasing with the tested dose. The inhibition zone of C. albicans grew with the increase in the volume of the extract, from 15 mm (50 µL) to 17 mm (75 µL) up to 20 mm (100 µL); the negative control DMSO showed no measurable inhibition beyond the 10-mm well diameter. The results once again showed that the concentration influences the zone of inhibition for A. flavus with the inhibition zones of 13 mm, 15 mm and 17 mm respectively at 50 µL, 75 µL and 100 µL, compared to the inhibition zone of 10 mm for the control. The dose–response relationship can be observed from the increasing diameter of inhibition zone with increasing concentration of the nanocomposite as the concentration will increase the fungal growth will be suppressed. The two fungal species showed different sensitivity towards the Ti₃C₂–Co₃(PO₄)₂ nanocomposite, with larger inhibition zone for C. albicans at the highest concentration. This increased susceptibility could relate to structural composition and/or permeability of the fungal cell wall and plasma membrane. The nanocomposite can be attributed to highly conductive Ti₃C₂ MXene nanosheets and bioactive Co₃(PO₄)₂ nanoparticles, which show effect when used together. The observed antifungal activity may result from complementary interactions between the Ti₃C₂ nanosheets and Co₃(PO₄)₂ nanoparticles. Surface-mediated interactions and the physicochemical properties of the two components may contribute to the observed inhibition; however, the specific mechanisms were not directly investigated. The nanosheet morphology may facilitate close interaction with fungal cell surfaces and could contribute to membrane-associated effects. The combined chemical and physical mechanisms are highly effective in preventing fungal growth and in lowering the viability of the cells. The lack of any significant inhibition in the control group also demonstrates that the antifungal activity is only due to the Ti₃C₂–Co₃(PO₄)₂ nanocomposite. In general, the synthesized nanocomposite shows promising concentration-dependent antifungal efficacy against yeast and filamentous fungi, indicating its potential as a multifunctional antimicrobial coating or biomaterial in various applications where it is crucial to prevent fungal colonization and biofilm formation, such as dental and biomedical applications and in healthcare.

Figure 6. Antifungal activity of Ti₃C₂–Co₃(PO₄)₂ nanocomposite against Candida albicans (a) and  Aspergillus flavus (b).

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite were measured by the standardized broth microdilution method, which enabled a quantitative measurement of the antifungal activity of the compound against Candida albicans and Aspergillus flavus. In brief, fungal inocula were adjusted to the desired cell density and inoculated in serial two-fold dilution of the nanocomposite in a sterile broth medium in sterile 96-well microplates. The positive control was fluconazole and the growth control, the medium sterility control and solvent control were included to validate the experimental procedure. MIC was defined as the lowest concentration of Ti₃C₂–Co₃(PO₄)₂ showing no visible fungal growth compared with the untreated growth control. MFC was defined as the lowest concentration that produced no visible fungal colony growth following subculture of aliquots from wells showing no visible growth. Aliquots from the wells where no growth was observed were transferred to fresh agar plates and the lowest concentration that did not yield fungal colony growth after incubation was considered the fungicidal concentration, which was then used to determine the MFC. Candida albicans was more susceptible to the Ti₃C₂–Co₃(PO₄)₂ nanocomposite with a MIC value of 62.5 µg/mL than A. flavus, which had a MIC value of 125 µg/mL. The MFC values were 125 µg/mL for C. albicans and 250 µg/mL for A. flavus. In contrast, the MICs of fluconazole for C. albicans and A. flavus were 16 and 32 µg/mL, respectively, indicating its well-established activity as a conventional antifungal agent. The MIC and MFC results demonstrate inhibitory and fungicidal activity against the tested fungal strains. Further, the MIC and MFC are comparable to those of the agar well diffusion method, which showed 20 mm inhibition zone for C. albicans and 17 mm for A. flavus at the highest tested concentration. The MIC and MFC results demonstrate measurable inhibitory and fungicidal activity against the tested fungal strains. The findings further demonstrate measurable antifungal activity of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite against the two tested fungal strains under the experimental conditions, supporting its potential for oral healthcare applications such as antimicrobial dental coatings, modification of implant surfaces and periodontal regenerative therapy.

Antioxidant and anti-inflammatory activity

The redox-modulatory performance of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite was systematically evaluated through DPPH radical scavenging, nitric oxide (NO) inhibition, and cytokine/ROS suppression assays. Results demonstrated concentration-dependent changes in DPPH scavenging, NO inhibition, cytokine levels and intracellular ROS were observed (Figure 7A–C).

Antioxidant activity (DPPH assay)

The antioxidant activity of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite is plotted against its concentration in the DPPH free radical scavenging assay as shown in Figure 7A, indicating that the nanocomposite has antioxidant activity in the range of concentrations studied (5–100 µg/mL). As the concentration of the nanocomposites increased, the percentage of DPPH radical inhibition increased gradually, DPPH radical-scavenging activity increased progressively with increasing nanocomposite concentration, from 14.33 ± 4.04% at 5 µg/mL to 28.67 ± 4.04%, 51.67 ± 4.51%, 72.67 ± 2.52% and 90.00 ± 2.00% at 10, 25, 50 and 100 µg/mL, respectively. The apparent DPPH IC₅₀, estimated by interpolation of the concentration-response data, was approximately 24 µg/mL, indicating the ability of this nanocomposite to scavenge DPPH free radicals. The observed concentration-dependent radical-scavenging response may be associated with the surface functional groups and physicochemical characteristics of the Ti₃C₂–Co₃(PO₄)₂ composite. However, specific redox couples, electron-transfer pathways and the individual contributions of Ti₃C₂ and Co₃(PO₄)₂ were not directly investigated in the present study. Such antioxidant activity of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite is thought to be due to the effect of the highly conductive Ti₃C₂ MXene nanosheets and the electrochemically active Co₃(PO₄)₂ nanoparticles. The large surface hydroxyl (–OH) and oxygen containing functional groups and terminal functionalities on the surface of Ti₃C₂ readily act as electron donors or hydrogen sources to reduce the DPPH radical, and the observed radical-scavenging activity may be associated with oxygen-containing surface functionalities of Ti₃C₂ and the chemical characteristics of the cobalt phosphate phase. The contribution of specific redox couples or electron-transfer pathways was not directly examined in the present study. Moreover, the large specific surface area of MXene sheets further facilitates the contact between the antioxidant active sites and DPPH molecules, which will facilitate the radical scavenging process. The close contact between Ti₃C₂ and Co₃(PO₄)₂ also may facilitate interfacial electron-transfer processes between them, leading to better redox efficiency of the composite. The relatively small standard deviations obtained at each concentration may be attributed to the high experimental reproducibility, dispersion stability of the nanocomposite and reliability of the antioxidant activity. The results show that the Ti₃C₂–Co₃(PO₄)₂ nanocomposite has antioxidant activity, and also acts as a good free radical scavenger.

Nitric oxide (NO) inhibition assay

To measure the inhibitory effect of Ti₃C₂–Co₃(PO₄)₂ nanocomposite, nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, a widely accepted in vitro model for assessment of inflammatory responses was used. The concentration-dependent inhibition of NO production was clearly observed from the presented results in the nanocomposite within the tested concentration range (5–100 µg/mL), as shown in Figure 7B. NO inhibition increased progressively from 12% at 5 µg/mL to 24%, 45%, 65% and 82% at 10, 25, 50 and 100 µg/mL, respectively. Higher concentrations of the nanocomposite produced greater suppression of NO production. The concentration-dependent reduction in NO production indicates attenuation of the inflammatory response under the tested conditions; however, direct effects on iNOS expression or activity were not investigated. The concentration corresponding to approximately 50% NO inhibition was estimated by interpolation to be approximately 31 µg/mL. The anti-inflammatory effect observed is probably due to the effect between the conductive Ti₃C₂ MXene nanosheets and the cobalt phosphate nanoparticles that work together to alleviate oxidative stress and regulate inflammatory signaling pathways via macrophages. The stability in results with low standard deviation values confirm the results were experimentally reproducible and show the potential of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite as an anti-inflammatory material for future biomedical and tissue engineering applications.

Cytokine and intracellular ROS suppression

To further assess the immunomodulatory effect of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite, the expression of the major pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), and the level of intracellular reactive oxygen species (ROS) were quantified as presented in Figure 7C. The available cytokine data showed a concentration-dependent decrease in TNF-α, IL-6 and IL-1β levels in a concentration-dependent manner in the results. TNF-α decreased from 85% of the untreated control at 5 µg/mL to 72%, 55%, 40% and 22% at 10, 25, 50 and 100 µg/mL, respectively. IL-6 decreased from 88% to 76%, 60%, 45% and 25%, while IL-1β decreased from 90% to 80%, 65%, 50% and 28% over the same concentration range. The simultaneous decrease in ROS generation and pro-inflammatory cytokine secretion could indicate that the Ti₃C₂–Co₃(PO₄)₂ nanocomposite acts on inflammation on several complementary levels. The simultaneous reduction in intracellular ROS and pro-inflammatory cytokines suggests that the nanocomposite may influence oxidative-stress-associated inflammatory responses. However, the specific molecular pathways responsible for these effects were not investigated. The observed reduction in intracellular ROS and inflammatory mediators may reflect attenuation of oxidative and inflammatory responses; however, the molecular pathways responsible were not directly investigated. The overall results showed that the Ti₃C₂–Co₃(PO₄)₂ nanocomposite has antioxidant and anti-inflammatory activity which can help in restoring the redox balance of cells, reducing overactive inflammatory responses, and shielding cells from oxidative damage. The multifunctional nature of the biological activities indicates that this material has promising biomedical applications, including oral tissue engineering, craniofacial regeneration, implant surface modification, and other therapeutic uses where the ability to effectively manage inflammation and oxidative stress is crucial for enhanced healing and clinical function. The observed reductions in NO, inflammatory cytokines and intracellular ROS indicate attenuation of inflammatory and oxidative responses under the tested conditions. However, the molecular pathways responsible for these effects were not directly investigated.

Figure 7. Antioxidant and anti-inflammatory responses of Ti₃C₂–Co₃(PO₄)₂ nanocomposite. DPPH radical-scavenging activity at 5–100 µg/mL; data are presented as mean ± SD based on three measurements (a). NO inhibition in LPS-stimulated RAW 264.7 macrophages at 5–100 µg/mL; values are presented descriptively based on the available dataset (b). TNF-α, IL-6, IL-1β and intracellular ROS levels following treatment with the nanocomposite; values are presented descriptively based on the available dataset (c).

pH stability and saliva simulation test

The environmental stability of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite was systematically studied under simulated oral conditions using Fusayama–Meyer artificial saliva to assess its potential for short-term stability under simulated salivary conditions in dental and biomedical fields (Figure 8). The nanocomposite retained 95 ± 1%, 100 ± 1% and 98 ± 1% of its initial weight after 48 h exposure to simulated saliva at pH 5.5, 6.5 and 7.5, respectively. No visible colour change, precipitation, aggregation or phase separation was observed during the exposure period. These observations indicate short-term physicochemical stability based on weight retention and visual assessment. However, post-exposure XRD or FTIR analyses were not performed; therefore, structural or phase stability after saliva exposure cannot be conclusively established.

Figure 8. pH stability of Ti₃C₂–Co₃(PO₄)₂ nanocomposite after 48 h immersion in simulated saliva at pH 5.5, 6.5, and 7.5 (37°C). Data are presented as mean ± SD (n = 3). Different lowercase letters indicate statistically significant differences among pH conditions according to one-way ANOVA followed by Tukey’s multiple-comparison test (P < 0.05).

MTT assay

Cell viability was evaluated using the MTT assay in RAW 264.7 macrophages exposed to 5–100 µg/mL Ti₃C₂–Co₃(PO₄)₂. Cell viability decreased gradually with increasing concentration, from 96.3 ± 2.8% at 5 µg/mL to 82.1 ± 3.4% at 100 µg/mL, compared with 100 ± 2.6% in the untreated control. All tested concentrations maintained cell viability above 80% under the experimental conditions. These findings indicate relatively low cytotoxicity in the RAW 264.7 macrophage model; however, they do not establish biocompatibility in normal oral cells.

Figure 9. MTT-based cell viability assessment of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite in RAW 264.7 macrophage cells. Cell viability was evaluated after exposure to Ti₃C₂–Co₃(PO₄)₂ at concentrations of 5, 10, 25, 50, and 100 µg/mL using the MTT assay. Data are presented as mean ± standard deviation (SD, n = 3).

DISCUSSION

The Ti₃C₂–Co₃(PO₄)₂ nanocomposite synthesized in this study displayed distinct structural and functional features directly linked to its multifunctional biological performance. The XRD pattern was consistent with the formation of Ti₃C₂ and Co₃(PO₄)₂ phases. XPS analysis was additionally used to assess the presence of residual Al following etching. The peak shifts toward lower 2θ values indicated interlayer expansion in Ti₃C₂, suggesting delamination and improved surface activation. The coexistence of hexagonal Ti₃C₂ and monoclinic Co₃(PO₄)₂ phases demonstrates a highly integrated interface that facilitates charge transfer between conductive and redox-active sites. The coexistence of Ti₃C₂ and Co₃(PO₄)₂ provides a physicochemically distinct composite interface that may contribute to the observed biological responses; however, the specific contribution of interfacial interactions was not directly investigated (Mao et al., 2018; Bakare et al., 2022).

FTIR spectroscopy confirmed strong interfacial bonding between both phases through characteristic vibrations corresponding to hydroxyl/surface oxygen-containing groups, phosphate and Co–O-related vibrations. The presence of O–H and P=O stretching bands indicates efficient electron delocalization and ionic stabilization at the composite interface. This bonding network enhances redox cycling and improves compatibility with aqueous and biological environments. Similar functional groups in phosphate-modified MXenes have been linked with improved antioxidant and antimicrobial potential due to hydroxyl–phosphate interactions. These features render the Ti₃C₂–Co₃(PO₄)₂ nanocomposite bio-interactive, enabling effective electron mediation and reactive oxygen species (ROS) regulation in physiologic conditions (Arunachalam et al., 2017; Li et al., 2017).

The UV–visible spectrum revealed dual absorption bands, one in the UV region (200–280 nm) corresponding to π–π* transitions in Ti₃C₂, and another broad visible band (400–750 nm) associated with d–d transitions of Co²⁺/Co³⁺. The broad optical response confirms strong electronic coupling between the two components, which may contribute to visible-light-driven activation and enhanced redox modulation. This optical property is advantageous in oral tissue applications, where controlled radical modulation can help mitigate oxidative stress-related disorders such as mucositis, peri-implantitis, and periodontitis (Tsujimoto et al., 2015; Yang et al., 2020).

Transmission electron microscopy confirmed the uniform dispersion of Co₃(PO₄)₂ nanoparticles within the Ti₃C₂ layers, with lattice spacings of 0.249 nm (Ti₃C₂, 006 plane) and 0.34 nm (Co₃(PO₄)₂, 141 plane), demonstrating high crystallinity and interfacial coherence. The polycrystalline nature observed in SAED patterns verifies strong integration and nanoscale ordering. This structural homogeneity increases the density of active sites and contributes to consistent biological activity. The resulting nanoarchitecture provides both mechanical stability and sustained functionality in oral environments, supporting its potential use in coatings and restorative materials (Su et al., 2014; Lim et al., 2015; Karunakaran, et al., 2026).

The antifungal analysis showed pronounced dose-dependent inhibition against Candida albicans and Aspergillus flavus. C. albicans displayed the highest susceptibility, with a 20 mm inhibition zone at 100 µL concentration, compared with 17 mm for A. flavus. This difference likely arises from variations in fungal cell wall structure and ergosterol content. The negatively charged Ti₃C₂ surface, the surface characteristics of Ti₃C₂ and the chemical properties of the cobalt phosphate phase may disrupt membrane integrity and metabolic pathways, leading to cell leakage and death. Such concentration-responsive inhibition indicates that the composite can prevent fungal colonization, suggesting its utility in denture bases, implant coatings, and restorative applications where Candida-related infections are common (Lim et al., 2020; Ahamed et al., 2022; Gowthami et al., 2023; Buanpech et al., 2024).

The DPPH assay confirmed strong antioxidant capacity, with radical scavenging activity increasing from 15% to 90% between 5 and 100 µg/mL. This reflects a redox interaction between Ti₃C₂ surface hydroxyl groups and Co²⁺/Co³⁺ centers in Co₃(PO₄)₂, enhancing electron or proton donation efficiency. As oxidative stress contributes to periodontal inflammation and delayed tissue healing, such free-radical neutralization is valuable in maintaining cellular viability and reducing oxidative injury in gingival or peri-implant tissues. The observed activity may be associated with oxygen-containing surface functionalities of Ti₃C₂ and the chemical characteristics of the cobalt phosphate phase; however, specific electron-transfer pathways were not directly investigated (Baliyan et al., 2022; Geng et al., 2022; Das and Saikia, 2023; Hammouda et al., 2024; Saravanan et al., 2025).

The anti-inflammatory evaluation using LPS-stimulated RAW 264.7 macrophages demonstrated substantial reductions in nitric oxide (NO) generation and pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. NO inhibition reached 82% at 100 µg/mL, accompanied by marked decreases in intracellular ROS levels. The reduction in NO, cytokines and intracellular ROS suggests attenuation of inflammatory and oxidative responses in LPS-stimulated macrophages. The specific signaling pathways responsible for these effects were not directly investigated. The dual antioxidative and anti-inflammatory response highlights its immunomodulatory nature, indicating promise for reducing peri-implant inflammation and soft-tissue irritation (Baek et al., 2020; Missier et al., 2023; Pal et al., 2023).

The pH stability and saliva simulation test further confirmed the nanocomposite’s physicochemical robustness under conditions mimicking the oral cavity. Across the pH range of 5.5–7.5, the stability index exceeded 95%, with no visible disintegration or phase alteration, indicating high tolerance to both acidic and neutral conditions. The observed stability may be associated with interfacial interactions between the Ti₃C₂ surface functionalities and the cobalt phosphate phase; however, the specific bonding contributions to salivary stability were not directly established. These features ensure short-term stability under simulated salivary conditions. These observations indicate short-term physicochemical stability under the tested simulated salivary conditions; longer-duration studies are required to establish stability during prolonged oral exposure (Raza et al., 2018; Ma et al., 2022). These findings support further investigation of the material in oral biomaterial systems, including surface-coating and related applications, following validation in relevant biofilm, normal oral-cell, long-term stability and in vivo models.

CONCLUSION

Ti₃C₂–Co₃(PO₄)₂ was successfully synthesized by a microwave-assisted method and exhibited measurable antifungal, antioxidant and anti-inflammatory responses under the tested in vitro conditions. The nanocomposite showed inhibition against C. albicans and A. flavus, DPPH radical-scavenging activity, concentration-dependent reductions in NO, inflammatory cytokines and intracellular ROS, and cell viability above 80% in RAW 264.7 macrophages. Weight retention of 95–100% was observed after 48 h exposure to simulated saliva over pH 5.5–7.5. These findings provide preliminary evidence of multifunctional in vitro biological activity and short-term physicochemical stability of the Ti₃C₂–Co₃(PO₄)₂ nanocomposite under the tested conditions. However, the absence of mature oral biofilm studies, normal oral-cell models, hemocompatibility testing, long-term degradation and cytotoxicity assessment, ion-release characterization, post-exposure structural analysis and in vivo validation limits conclusions regarding dental application. Further studies are required before the material can be considered for specific clinical or dental uses.

ACKNOWLEDGEMENTS

The authors would like to thank the Department of Physiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India, for providing research laboratory facilities for conducting the study and providing support to complete the work successfully.

AUTHOR CONTRIBUTIONS

Daivik Gupta Majety: Data Curation (Equal), Formal Analysis (Equal), Writing – Original Draft (Equal), Writing – Review & Editing (Equal), Investigation (Lead); Geetha Anbalagan: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Lead), Validation (Lead), Resource (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Lead), Investigation (Equal), Supervision (Lead), Project Administration (Equal);  Ilangovar Indirani Ganesan Kannan: Methodology (Supporting), Formal Analysis (Supporting), Validation (Equal), Resource (Lead), Data Curation (Lead), Writing – Review & Editing (Equal), Investigation (Supporting), Supervision (Equal), Project Administration (Supporting); Vasugi Suresh: Data Curation (Equal), Formal Analysis (Equal), Writing – Original Draft (Equal), Investigation (Lead); Balachandran Subramanian: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Equal), Validation (Lead), Resource (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Lead), Investigation (Equal), Supervision (Lead), Project Administration (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

REFERENCES

Ahamed, M., Akhtar, M.J., Khan, M.A.M., and Karuppiah, P. 2022. Antibacterial, antifungal, and anticancer potential of two-dimensional Ti₃C₂Tₓ MXene. Materials Letters. 327: 133020. https://doi.org/10.1016/j.matlet.2022.133020

Amann, V., Kissmann, A.-K., Firacative, C., and Rosenau, F. 2025. Biofilm-associated candidiasis: Pathogenesis, prevalence, challenges and therapeutic options. Pharmaceuticals. 18(4): 460. https://doi.org/10.3390/ph18040460

Anwar, A., Chi Fung, L., Anwar, A., Jagadish, P., Numan, A., Khalid, M., Shahabuddin, S., Siddiqui, R., and Khan, N.A. 2019. Effects of shape and size of cobalt phosphate nanoparticles against Acanthamoeba castellanii. Pathogens. 8(4): 260. https://doi.org/10.3390/pathogens8040260

Arunachalam, P., Shaddad, M.N., Alamoudi, A.S., Ghanem, M.A., and Al-Mayouf, A.M. 2017. Microwave-assisted synthesis of Co₃(PO₄)₂ nanospheres for electrocatalytic oxidation of methanol in alkaline media. Catalysts. 7(4): 119. https://doi.org/10.3390/catal7040119

Baek, S.-H., Park, T., Kang, M.-G., and Park, D. 2020. Anti-inflammatory activity and ROS regulation effect of sinapaldehyde in LPS-stimulated RAW 264.7 macrophages. Molecules. 25(18): 4089. https://doi.org/10.3390/molecules25184089

Bakare, T.E., Akintemi, E.O., Ogundare, S.A., and van Zyl, W.E. 2022. The Co₃O₄-Co₃(PO₄)₂ nanocomposite supercapacitor system: Synthesis, electrochemistry, temperature effects, and computational studies. Solid State Sciences. 131: 106951. https://doi.org/10.1016/j.solidstatesciences.2022.106951

Baliyan, S., Mukherjee, R., Priyadarshini, A., Vibhuti, A., Gupta, A., Pandey, R.P., and Chang, C.-M. 2022. Determination of antioxidants by DPPH radical scavenging activity and quantitative phytochemical analysis of Ficus religiosa. Molecules. 27(4): 1326. https://doi.org/10.3390/molecules27041326

Buanpech, P., Chaijareenont, P., Wanachantararak, P., and Silthampitag, P. 2024. Antifungal effect and durability of chitosan oligosaccharide coating on heat-cured polymethylmethacrylate surface. Natural and Life Sciences Communications. 23(1): e2024004. https://doi.org/10.12982/NLSC.2024.004

Das, D. and Saikia, B.J. 2023. Synthesis, characterization and biological applications of cobalt oxide (Co₃O₄) nanoparticles. Chemical Physics Impact. 6: 100137. https://doi.org/10.1016/j.chphi.2022.100137

Desai, S.S., Manogaran, Y., Duraisamy, R., Ganapathy, D., and Ramasamy, P. 2026. Biogenic sulphated chitosan - vanadium pentoxide complexes from Sepiella inermis: A potential antimicrobial agent for oral pathogens. Natural and Life Sciences Communications. 25(3): e2026043. https://doi.org/10.12982/NLSC.2026.043

Devanshi, C., Geetha, A., Ilangovar, I.G.K., Vasugi, S., and Balachandran, S. 2024. Graphene- functionalized titanium carbide synthesis and characterization and its cytotoxic effect on cancer cell lines. Cureus. 16(5): e61049. https://doi.org/10.7759/cureus.61049

Geng, H., Ren, Y., Qin, G., Wen, T., Liu, Q., Xu, H., and He, W. 2022. Ti₃C₂ nanosheets with broad-spectrum antioxidant activity for cytoprotection against oxidative stress. RSC Advances. 12(18): 11128-11138. https://doi.org/10.1039/D2RA01225A

Gowthami, P., Kosiha, A., Meenakshi, S., Boopathy, G., Ramu, A.G., and Choi, D. 2023. Biosynthesis of Co₃O₄ nanomedicine by using Mollugo oppositifolia L. aqueous leaf extract and its antimicrobial, mosquito larvicidal activities. Scientific Reports. 13(1): 9002. https://doi.org/10.1038/s41598-023-35877-z

Hadia, N.M.A., Karim, M.R., Alzaid, M., Hasaneen, M.F., Ezzeldien, M., Mohamed, W.S., Shafei, L., Mumtaz, S., Hassan, H., Iqbal, M.W., and Ali, S.K. 2025. Interfacial engineering of titanium carbide (Ti₃C₂Tₓ) MXene and samarium cobalt phosphate (SmCoPO₄) nanostructures boosts charge storage in hybrid supercapacitors. Journal of Inorganic and Organometallic Polymers and Materials. 36(5): 2678-2690. https://doi.org/10.1007/s10904-025-04053-3

Hammouda, M.M., Alanazi, A.A., and Elattar, K.M. 2024. Mentha suaveolens leaves extract- mediated synthesis of CoO@TiO₂ and Fe₂O₃@TiO₂ core/shell nanoparticles with antimicrobial and antioxidant activities. ChemistrySelect. 9(17): e202401385. https://doi.org/10.1002/slct.202401385

Harini, C.P., Geetha, A., Ilangovar, I.G.K., Vasugi, S., and Balachandran, S. 2024. Synthesis and application of titanium carbide (Ti₃C₂)-cobalt sulfide (Co₃S₄) nanocomposites in amino acid biosensing. Cureus. 16(7): e63582. https://doi.org/10.7759/cureus.63582

Kalaivani, V., Balasubramaniam, A., and Meignana Arumugham, I. 2025. Oral health status, self-assessment and risk among tribes and Narikuravars of Villupuram district, Tamil Nadu - An epidemiological study. Journal of Oral Biology and Craniofacial Research. 15(6): 1361-1366. https://doi.org/10.1016/j.jobcr.2025.08.018

Karunakaran, S., Pitchai, A., Duraisamy, R., Ganapathy, D., and Ramasamy, P. 2026. Characterization and in vitro anti-oral cancer potential of nanochitosan from cuttlebone of Sepia pharaonis (Ehrenberg, 1831). Tanta Dental Journal. 23(1): 33-39. https://doi.org/10.4103/tdj.tdj_99_25

Kashyap, B., Padala, S.R., Kaur, G., and Kullaa, A. 2024. Candida albicans induces oral microbial dysbiosis and promotes oral diseases. Microorganisms. 12(11): 2138. https://doi.org/10.3390/microorganisms12112138

Lee, Y., Puumala, E., Robbins, N., and Cowen, L.E. 2021. Antifungal drug resistance: Molecular mechanisms in Candida albicans and beyond. Chemical Reviews. 121(6): 3390- 3411. https://doi.org/10.1021/acs.chemrev.0c00199

Li, Y., Zhou, Y., Wang, X., Deng, J., Li, Q., Du, M., Han, S., Lee, Y.-H., Jaehyung, J., and Huang, Q. 2017. Facile preparation of in situ coated Ti₃C₂Tₓ/Ni₀.₅Zn₀.₅Fe₂O₄ composites and their electromagnetic performance. RSC Advances. 7(40): 24698-24708. https://doi.org/10.1039/C7RA03402D

Lim, G.P., Soon, C.F., Morsin, M., Ahmad, M.K., Nayan, N., and Tee, K.S. 2020. Synthesis, characterization and antifungal property of Ti₃C₂Tₓ MXene nanosheets. Ceramics International. 46(12): 20306-20312. https://doi.org/10.1016/j.ceramint.2020.05.118

Lim, S., Pandikumar, A., Lim, H., Ramaraj, R., and Huang, N. 2015. Boosting photovoltaic performance of dye-sensitized solar cells using silver nanoparticle-decorated N,S-co-doped TiO₂ photoanode. Scientific Reports. 5: 11922. https://doi.org/10.1038/srep11922

Ma, T., Wang, X., and Chen, J. 2022. In vitro stability study of saliva emulsions: The impact of time, calcium ion and pH. Food Hydrocolloids. 125: 107390. https://doi.org/10.1016/j.foodhyd.2021.107390

Mao, H., Zhang, F., Liu, X., Qiu, J., Li, B., and Jin, Z. 2018. Synthesis of cobalt phosphate nanoflakes for high-performance flexible symmetric supercapacitors. Journal of Materials Science: Materials in Electronics. 29(19): 16721-16729. https://doi.org/10.1007/s10854-018-9765-x

Missier, M.S., Ramakrishnan, M., Sudalaimani Paulpandian, S.D., Rajeshkumar, S., and Tania, M. 2023. Antibacterial, antioxidant and anti-inflammatory activity of zinc-titanium dioxide nanocomposite. Bioinformation. 19(5): 638-643. https://doi.org/10.6026/97320630019638

Pal, P.P., Begum, A.S., Basha, S.A., Araya, H., and Fujimoto, Y. 2023. New natural pro- inflammatory cytokines (TNF-α, IL-6, and IL-1β) and iNOS inhibitors identified from Penicillium polonicum through in vitro and in vivo studies. International Immunopharmacology. 117: 109940. https://doi.org/10.1016/j.intimp.2023.109940

Rajasri, S., Kalikeri, S., Balachandran, S., Gowthami, K., Thirunarayanan, G., Swaminathan, M., and Muthuvel, I. 2022. Enhanced photodegradation of 2,4-dinitrophenol by n-p type TiO₂/BiOI nanocomposite. Journal of the Indian Chemical Society. 99(3): 100337. https://doi.org/10.1016/j.jics.2021.100337

Raza, T., Andleeb, S., Ullah, S., Jamal, M., Mehmood, K., and Ali, M. 2018. Isolation and characterization of a phage to control vancomycin-resistant Enterococcus faecium. Open Life Sciences. 13(1): 553-560. https://doi.org/10.1515/biol-2018-0066

Saravanan, M., Mahalakshmi, B., Afeeza, K.L.G., Subriya, S., Sridevi, M., and Dilipan, E. 2025. Formulation and characterization of sodium alginate-based nanoparticle doped with seagrass (Syringodium isoetifolium) extract: In vitro anti-inflammatory, antioxidant and antidiabetic activities. Natural and Life Sciences Communications. 24(2): e2025035. https://doi.org/10.12982/NLSC.2025.035

Su, D., Dou, S., and Wang, G. 2014. Single crystalline Co₃O₄ nanocrystals exposed with different crystal planes for Li-O₂ batteries. Scientific Reports. 4: 5767. https://doi.org/10.1038/srep05767

Tamhane, O., Geetha, A., Vasugi, S., Balachandran, S., and Ilangovar, I.G.K. 2024. Optimising the synthesis of titanium carbide-bismuth oxide for enhanced antimicrobial properties. Cureus. 16(8): e67971. https://doi.org/10.7759/cureus.67971

Thongjan, N., Prapamontol, T., Promkam, N., Somsunun, K., Naksen, W., and Norbäck, D. 2025. Association between urinary metabolites of organophosphate insecticides and malondialdehyde oxidative stress biomarker among adults in a rural area of Chiang Mai, Thailand: A follow-up study in two post-harvest seasons. Natural and Life Sciences Communications. 24(2): e2025030. https://doi.org/10.12982/NLSC.2025.030

Tsujimoto, Y., Matsushita, Y., Yu, S., Yamaura, K., and Uchikoshi, T. 2015. Size dependence of structural, magnetic, and electrical properties in corundum-type Ti₂O₃ nanoparticles showing insulator-metal transition. Journal of Asian Ceramic Societies. 3(3): 325-333. https://doi.org/10.1016/j.jascer.2015.06.007

Yang, P., Li, W., Lian, Y., Yu, F., Dai, B., Guo, X., Liu, Z., and Peng, B. 2020. A facile approach to synthesize CoO-Co₃O₄/TiO₂ NAs for reinforced photoelectrocatalytic water oxidation. Journal of Solid State Electrochemistry. 24: 941-950. https://doi.org/10.1007/s10008-020-04528-y         

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