Doxorubicin-Loaded Cerium Oxide Nanoparticles Suppress Cell Proliferation, Migration and Invasion of Triple-Negative Breast Cancer Cells

Abstract

Cerium oxide nanoparticles (Ceria NPs), possessing redox activity, exhibit selective anticancer effects and hold significant promise for cancer therapy. In this study, Ceria NPs were synthesized via a hydrothermal method and characterized for stability in a biological environment. Optimal colloidal dispersion was achieved in 10% fetal bovine serum (FBS) with a hydrodynamic diameter of 56.3 nm. Ceria NPs exhibited strong antioxidant activity tested by DPPH assay and showed high biocompatibility, displaying minimal toxicity toward normal breast epithelial cells (MCF-10A) while exerting cytotoxic effects on breast cancer cells (IC₅₀ = 363.0 ± 71.89 μg mL^–1 for MCF-7 and 547.9 ± 0.14 μg mL^–1 for MDA-MB-231). Given their limited standalone cytotoxicity but favorable selectivity, Ceria NPs were employed as a nanocarrier for doxorubicin (Dox) to enhance anticancer efficacy while potentially reducing systemic side effects. Ceria-Dox markedly enhanced cytotoxic efficacy, reducing IC₅₀ values to 129.3 ± 21.86 μg mL^–1 in MCF-7 and 33.2 ± 1.37 μg mL^–1 in MDA-MB-231 cells, with more pronounced effects in TNBC cells while maintaining low toxicity toward normal cells. Live/dead staining confirmed a dose-dependent cytotoxicity, while the transwell assay demonstrated strong inhibition of migration and invasion in TNBC cells treated with Ceria-Dox. These findings highlight Ceria-Dox as a colloidally stable nanoplatform that delivers dual therapeutic actions, including potent cytotoxicity and suppression of metastatic potential, underscoring its promise for treating breast cancer, particularly aggressive subtypes such as triple-negative breast cancer.

1. Introduction

Breast cancer is one of the most prevalent types of cancer, and triple-negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer. − TNBC is defined by the absence of estrogen receptor, progesterone receptor, and HER2, limiting targeted treatment options and contributing to poor clinical outcomes, high recurrence, and increased metastatic potential. Despite advances in chemotherapy, surgery, and radiotherapy, conventional treatments remain limited by systemic toxicity, nonspecific drug distribution, and the development of multidrug resistance. , Therefore, the development of selective and effective therapeutic strategies is urgently needed.

Nanotechnology-enabled drug delivery platforms offer a promising strategy to enhance therapeutic efficacy while reducing off-target toxicity. A wide range of nanomaterials, including polymeric nanospheres, gold nanoparticles, quantum dots, and magnetic nanoparticles, have been developed to deliver diverse therapeutic agents such as hydrophilic or hydrophobic drugs, vaccines, and biological macromolecules. Among these, cerium oxide nanoparticles, or nanoceria (Ceria NPs), have attracted attention due to their unique redox-active Ce³⁺/Ce⁴⁺ cycling, which allows precise modulation of reactive oxygen species (ROS). − This redox flexibility enables CeO₂ NPs to modulate ROS in a microenvironment-dependent manner, functioning as antioxidants under physiological conditions while exhibiting pro-oxidant activity in tumor environments.

Such dual redox behavior is particularly advantageous for cancer therapy. In normal cells, CeO₂ NPs can scavenge ROS and maintain redox homeostasis, contributing to their biocompatibility. In contrast, the acidic and oxidative tumor microenvironment promotes a shift toward pro-oxidant activity, leading to increased intracellular ROS, oxidative stress, and cancer cell death. Additionally, partial dissolution of CeO₂ NPs under acidic conditions facilitates Ce³⁺ release, further enhancing redox-mediated cytotoxicity. These features make CeO₂ NPs attractive candidates for selective cancer therapy. , However, their relatively limited standalone cytotoxicity necessitates further functionalization to improve therapeutic outcomes.

Doxorubicin (Dox), a widely used anthracycline chemotherapeutic, exhibits potent anticancer activity through DNA intercalation and inhibition of topoisomerase II. , However, Dox is limited by poor selectivity, dose-limiting toxicity, particularly cardiotoxicity, and the emergence of multidrug resistance. , To overcome these limitations, nanocarrier-based delivery approaches have been studied to improve tumor-specific accumulation while minimizing systemic toxicity. In this context, CeO₂ NPs offer a multifunctional platform for drug delivery. Loading Dox onto CeO₂ NPs can improve drug stability, enable controlled release, and enhance intracellular accumulation. Furthermore, the combination of Dox-induced DNA damage with CeO₂-mediated ROS modulation may produce synergistic anticancer effects, improving efficacy while potentially reducing adverse effects. ,

In this study, we developed Ceria-Dox for breast cancer therapy, focusing on its colloidal stability in biologically relevant media and the therapeutic enhancement conferred by drug loading. The system was evaluated for selective cytotoxicity against breast cancer cells and its potential to inhibit metastatic behaviors, with particular attention to TNBC. This work demonstrates the potential of combining redox-active nanomaterials with chemotherapeutic drugs to improve tumor selectivity, reduce systemic toxicity, and overcome limitations associated with conventional chemotherapy.

2. Experimental Section

2.1. Materials

Cerium (III) nitrate hexahydrate (Ce­(NO₃)₃·6H₂O; 99%), and ethylene glycol were obtained from Sigma-Aldrich (Burlington, MA, USA). Sodium hydroxide anhydrous 98% and acetic acid were obtained from Carlo Erba (Milan, Italy). Ethyl alcohol was purchased from Duksan (Ansan, South Korea). The free radical scavenging reagent 2,2-diphenyl-1-picrylhydrazyl (DPPH), along with dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagents, insulin, and hydrocortisone were obtained from Sigma-Aldrich (Burlington, MA, USA). Doxorubicin hydrochloride (Dox), Dulbecco’s Modified Eagle Medium (DMEM), Eagle’s Minimum Essential Medium (EMEM), DMEM/F12, penicillin–streptomycin, fetal bovine serum (FBS), horse serum, and human epidermal growth factor (hEGF) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). All reagents and chemicals were analytical grade and used as received.

2.2. Synthesis of Cerium Oxide Nanoparticles (Ceria NPs)

Ceria NPs were prepared following a previous report by Mai et al. with minor modifications. In brief, 1.3020 g of Ce­(NO₃)₃·6H₂O was dissolved in 45 mL of deionized water. The solution was adjusted to pH 10 using 2.5 M NaOH and continuously stirred for 96 h, then centrifuged at 25 °C and 16,099g for 15 min. Deionized water (35 mL) was used to wash the precipitate 5 times until the wash solution reached neutral pH (∼7), then the precipitate was washed with 35 mL of ethanol. The obtained solid was dried in a hot-air oven at 60 °C for 24 h, then calcined in a muffle furnace at 400 °C for 4 h.

2.3. Characterization of Ceria NPs

2.3.1. Scanning Electron Microscopy (SEM)

SEM image of Ceria NPs sample was investigated on FEI model Quanta450 SEM at an acceleration voltage of 25.00 kV.

2.3.2. Transmission Electron Microscopy (TEM)

TEM images were acquired using a JEOL JEM-2010 TEM operated at 120–200 kV. The NPs were redispersed at 0.5 mg mL^–1 in ethanol before being added onto a carbon-supported copper TEM grid (PELCO, Ted Pella) and dried under ambient conditions prior to analysis.

2.3.3. Powder X-ray Diffraction (XRD)

XRD analysis was performed to determine the crystalline structure of the synthesized CeO₂ nanoparticles. Diffraction patterns were recorded over a 2θ range of 5°–90° using a benchtop X-ray diffractometer (D8 Advance, Bruker, Germany) operated at 40 kV and 40 mA with Cu Kα radiation (λ = 1.5418 Å).

2.3.4. X-ray Photoelectron Spectroscopy (XPS)

XPS was used to analyze the chemical composition of the CeO₂ nanoparticles. Measurements were carried out using a PHI 5000 VersaProbe II (ULVAC-PHI, Japan) at the SUT–NANOTEC–SLRI Joint Research Facility, Synchrotron Light Research Institute (SLRI), Thailand. A monochromatized Al Kα X-ray source (1486.6 eV) was employed as the excitation source. The obtained spectra were subsequently deconvoluted and fitted using Gaussian–Lorentzian functions.

2.3.5. Fourier Transform Infrared Spectroscopy Analysis (FTIR)

FTIR spectroscopy was conducted to examine the vibrational features of both the synthesized CeO₂ nanoparticles and CeO₂–Dox nanoparticles, as well as to confirm the presence of functional groups. The spectra were collected using a Bruker Tensor 27 FTIR spectrometer over a wavenumber range of 4000–500 cm^–1, with 32 scans and a resolution of 4 cm^–1. This analysis was performed to detect characteristic absorption peaks associated with the nanoparticles’ chemical structure, bonding interactions, and phase stability.

2.4. The Colloidal Stability of Ceria NPs

The dispersion behavior of Ceria NPs and Ceria-Dox under physiological conditions was studied using hydrodynamic diameter and zeta potential measurements. Suspensions were prepared at 10 μg mL^–1 in deionized water, 1× PBS (pH 7.4), and DMEM containing 1%, 5%, and 10% heat-inactivated fetal bovine serum (FBS) to simulate different biological environments. To evaluate nanoparticle stability in biological environments, the hydrodynamic diameter (DH), polydispersity index (PdI), and zeta potential (ζ) were measured at 25 °C using a Zetasizer Ultra Red (Malvern Instruments, UK). These parameters were used to assess particle size distribution, aggregation tendency, and surface charge.

2.5. DPPH Radical Scavenging Assay

A DPPH (1,1-diphenyl-2-picrylhydrazyl) working solution (0.1 mM) was freshly prepared in ethanol and protected from light. Various concentrations of Ceria NPs were prepared in ethanol. To evaluate DPPH radical scavenging activity, 100 μL of each sample solution was mixed with an equal volume of DPPH working solution in a 96-well plate. The reaction mixture was incubated at room temperature for 30 min in the dark. After incubation, the absorbance was measured at 517 nm using a microplate reader (Multiskan SkyHigh, Thermo Scientific). Trolox was used as the positive control, while ethanol served as the blank. The percentage of radical scavenging activity was calculated using the equation below

$$\%\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}=\frac{{A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-A}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

where A _control represents the absorbance of the DPPH solution without nanoparticles, and A _sample is the absorbance of the reaction mixture containing the nanoparticles.

2.6. Doxorubicin-Loaded Ceria NPs (Ceria-Dox)

To load Dox onto Ceria NPs, 500 μM Dox in PBS (pH 7.4) was mixed with Ceria NPs (1 mg mL^–1) and incubated under continuous stirring for 16 h in the dark using a similar method that was previously published. After incubation, the mixture was centrifuged at 15,000g for 10 min using a Multifuge X Pro Series ultracentrifuge (Thermo Scientific). The supernatant was collected to determine the drug-loading efficiency (% LE) using a UV–Vis spectrophotometer (Shimadzu) over the wavelength range of 350–600 nm. Unbound doxorubicin (Dox) in the supernatant was quantified at its maximum absorbance of 494 nm. The resulting pellet, corresponding to Dox-loaded Ceria nanoparticles (Ceria–Dox), was collected, dried at 50 °C, and stored at room temperature. Drug-loading efficiency was calculated according to the equation below

$$\%\mathrm{L}\mathrm{E}=\frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}-\mathrm{U}\mathrm{n}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{e}\mathrm{d}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}\times 100$$

2.7. In Vitro Drug Release Profile

Ceria-Dox was prepared at 4 mg mL^–1 in 1xPBS (pH 7.4), while free doxorubicin (Dox) was used at 200 μg mL^–1. The drug release study was performed using a dialysis membrane (M.W. cut off at 12,000–14,000 Da) in 5 mL PBS and incubated at 37 °C with continuous shaking at 200 rpm for 3 days. At each time point, 1 mL of the medium was collected and replaced with fresh PBS. Dox release was analyzed using fluorescence spectroscopy. The cumulative drug release (CDR) percentage of Dox released was calculated following the equation below

$$\%\mathrm{C}\mathrm{D}\mathrm{R}=\frac{\mathrm{D}\mathrm{o}\mathrm{x}\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{p}\mathrm{o}\mathrm{i}\mathrm{n}\mathrm{t}}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{D}\mathrm{o}\mathrm{x}}\times 100$$

The in vitro release kinetics of Dox-loaded Ceria nanoparticles was analyzed by fitting the drug release profiles to mathematical models using DDSolver, a Microsoft Excel add-in.

2.8. Cell Lines and Culture Conditions

The human breast cancer cell lines MDA-MB-231 (HTB-26) and MCF-7 (HTB-22), along with the normal mammary epithelial cell line MCF-10A (CRL-10317), were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231 and MCF-7 cells were cultured in DMEM and EMEM, respectively, both supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S). In contrast, MCF-10A cells were maintained in DMEM/F12 medium supplemented with 5% horse serum, insulin (10 μg mL^–1), hydrocortisone (0.5 μg mL^–1), human epidermal growth factor (hEGF; 20 ng mL^–1), and 1% P/S. All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

2.9. Cytotoxic Assay

The cytotoxicity of Ceria NPs or Ceria-Dox was evaluated using the MTT assay. Cells were seeded into 96-well plates at a density of 8 × 10³ cells per well and incubated overnight at 37 °C and 5% CO₂ to allow attachment. Subsequently, the cells were exposed to nanoparticles at 12.5, 25, 50, 100, 200, and 400 μg mL^–1 in complete medium and incubated for 48 h. After treatment, 0.5 mg mL^–1 MTT solution was added, and the mixture was incubated for an additional 4 h at 37 °C. The resulting formazan crystals were dissolved using 100 μL of dimethyl sulfoxide (DMSO) before measuring absorbance at 570 nm using a microplate reader (Multiskan SkyHigh, Thermo Scientific). Cell viability (%) was calculated relative to untreated control cells according to the following equation

$$\%\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{A_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

where A _sample and A _control represent the absorbance of treated and untreated cells, respectively.

2.10. Live-Dead Cells Assay

Live and dead cells were assessed using Calcein-AM and propidium iodide (PI) staining. Cells were seeded into black 96-well plates at a density of 8 × 10³ cells per well and incubated overnight at 37 °C in a humidified 5% CO₂. After treatment with 12.5, 25, and 50 μg mL^–1 of Ceria–Dox for 48 h, the cells were incubated with a staining solution containing 1 μM Calcein-AM (Invitrogen) and PI (1:200 dilution; Dojindo) prepared in complete medium. Following 30 min incubation at 37 °C in the dark, fluorescent images were immediately captured using an Operetta High-Content Imaging System (PerkinElmer, Waltham, MA, USA).

2.11. Intracellular ROS and Mitochondrial Membrane Potential Assays

Intracellular ROS production was measured using a DCFDA Cellular ROS Assay Kit (Abcam, ab113851) according to the manufacturer’s protocol. While mitochondrial membrane potential (MMP) was assessed using a JC-1 assay kit (Dojindo Laboratories, Japan), following the manufacturer’s protocol. The details of the assay procedures can be found in the Supporting Information methods.

2.12. Colony Formation Assay

MDA-MB-231 cells were seeded into 24-well plates at a density of 150 cells per well and treated with 10, 30, and 50 μg mL^–1 Ceria-Dox for 7 days. Following treatment, the cells were fixed with absolute methanol for 15 min and stained with 0.5% crystal violet for 30 min. The wells were then washed with distilled water and air-dried before imaging. The number of colonies formed was quantified using ImageJ software. Images were captured using an APX100 inverted fluorescence microscope (Olympus, Japan). All experiments were performed in triplicate.

2.13. Migration and Invasion Studies

MDA-MB-231 cells were seeded in a 12-well plate (1 × 10⁵ cells/well) and allowed to adhere for 24 h. Subsequently, cells were treated with Ceria-Dox at various concentrations (12.5, 25, and 50 μg mL^–1) and incubated for an additional 24 h. For the invasion part, transwell inserts were coated with Matrigel as an extracellular matrix (ECM). Then, cells were trypsinized, seeded again into the 24-well Transwell Permeable Support (Corning) chambers at 25,000 cells/well, and incubated for 24 h. After incubation, cells were fixed with 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet for 1 h. The nonmigrating cells in the upper chamber were carefully removed by cotton swabs. The image was captured using a light microscope at 10× magnification across 5 fields per well. Migrating and invading cells were quantified using ImageJ software (NIH, USA) and calculated as a percentage compared to the control condition.

2.14. Statistical Analysis

Data are presented as mean ± standard error of the mean (S.E.M.) from at least three independent experiments. Statistical analysis was performed using GraphPad Prism version 10 (GraphPad Software Inc., CA, USA). Differences among multiple groups were evaluated using one-way analysis of variance (ANOVA), and statistical significance was defined according to the criteria stated below: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05).

3. Results and Discussions

3.1. Morphological and Structural Characterization of Ceria NPs

Ceria NPs were successfully synthesized via the hydrothermal method and are presented in scanning electron microscopy (SEM, Figure a) and transmission electron microscopy (TEM, Figure b) analyses, which confirmed that the particles exhibited an irregular morphology. As shown in Figure c, X-ray photoelectron spectroscopy (XPS) further revealed the coexistence of cerium in mixed oxidation states (Ce³⁺/Ce⁴⁺). The deconvoluted peaks were assigned to Ce³⁺ (3d₅/₂ at 885 eV and 3d₃/₂ at 903 eV) and Ce⁴⁺ (3d₅/₂ at 882, 888, 898 eV and 3d₃/₂ at 900, 907, 916.5 eV). The O 1s spectrum (Figure d) showed two main peaks: one at 529–529.5 eV corresponding to lattice oxygen and another at 531–532 eV attributed to surface oxygen species or vacancy-related defects. XPS techniques confirmed the coexistence of 21.60% Ce³⁺ and Ce⁴⁺ valence states along with 10.99% oxygen vacancies (defect ¹O₂), indicating a high proportion of Ce³⁺ species and defect sites in NPs. These structural characteristics are critical because reversible Ce³⁺/Ce⁴⁺ redox cycling facilitates electron transfer processes that neutralize ROS (e.g., H₂O₂). This feature underlies the strong free-radical-scavenging activity of Ceria NPs, as confirmed by the antioxidant assay below. In addition, the Powder X-ray diffraction (XRD) pattern showed characteristic Bragg peaks indexed to the (111), (200), (220), (311), (222), (400), (331), and (420) planes, consistent with the cubic fluorite structure of Ceria NPs (Figure e). Collectively, these results confirm the successful synthesis of structurally well-defined CeO₂ nanoparticles with abundant Ce³⁺/Ce⁴⁺ redox-active sites and oxygen vacancies.

1.

Characterization of Ceria NPs. (a) SEM image showing particle morphology, Scale bar = 400 nm. (b) TEM image revealing detailed structure, Scale bar = 20 nm. (c) XPS analysis of surface composition. (d) XPS-derived evaluation of oxygen vacancy. (e) XRD pattern indicating crystalline structure.

3.2. Colloidal Dispersion Characteristics of Ceria NPs in Various Media

For the effective application of Ceria NPs in biological systems, evaluating their dispersion behavior in various dispersants is essential, as colloidal stability directly influences bioavailability, cellular uptake, and therapeutic performance. In physiological environments, nanoparticles interact with a complex mixture of ions, proteins, and biomolecules, leading to the formation of a protein corona that can alter surface charge, hydrodynamic size, and aggregation state. − Accordingly, the dispersion behavior of Ceria NPs was investigated by determining their hydrodynamic diameter and zeta potential in biologically relevant media, including water, PBS, and culture media containing 1%, 5%, and 10% FBS. The results from dynamic light scattering (DLS) measurements indicated that the hydrodynamic sizes of Ceria NPs varied with the dispersion medium. In water, the size was 460.5 ± 277.3 nm, whereas in PBS (pH 7.4) it increased to 942.2 ± 88.5 nm. The smallest size was observed in DMEM medium supplemented with 10% FBS, with an average hydrodynamic diameter of 56.3 ± 30.1 nm and a polydispersity index (PdI) of 0.41 ± 0.11 (Table ). These results suggest that the culture medium supplemented with 10% FBS effectively stabilized Ceria NPs, as indicated by the reduction in hydrodynamic diameter, due to the serum proteins play a crucial role in determining their colloidal stability, as previously reported in other NPs. Numerous studies have demonstrated that nanoparticles readily interact with surrounding proteins to form a protein corona, which can prevent aggregation and enhance dispersion stability. To further evaluate the colloidal stability of Ceria NPs under biologically relevant conditions, time-dependent stability studies were performed over 72 h, including hydrodynamic size analysis (Supporting Information Figure S1a), polydispersity index (PDI) measurements (Supporting Information Figure S1b), and zeta potential analysis (Supporting Information Figure S1c). These observations are consistent with our previous studies on superparamagnetic iron oxide nanoparticles (SPIONs), where substantial aggregation in water was significantly reduced in DMEM-containing medium, highlighting the critical role of the dispersing medium in nanoparticle stability and biological behavior. , Similarly, Ceria NPs exhibited optimal dispersion stability in 10% FBS-containing medium, which closely mimics physiologically relevant conditions. Therefore, all subsequent biological experiments were conducted using nanoparticles dispersed in 10% FBS-containing medium to ensure stable, reproducible, and biologically relevant experimental conditions.

1. Size, Polydispersity Index (PdI), and Zeta Potential of Ceria NPs in Different Biological Media Following 10 min of Ultrasonication .

biological medium	size (nm)	PdI	zeta potential (mV)
water	460.5 ± 277.3	0.49 ± 0.22	–13.82
PBS	942.2 ± 88.5	0.75 ± 0.04	–24.02
1% FBS	356.2 ± 163.2	0.43 ± 0.05	–11.43
5% FBS	174.7 ± 117.0	0.64 ± 0.26	–20.80
10% FBS	56.3 ± 30.1	0.41 ± 0.11	–10.35

^aData are presented as mean ± SEM (n = 3).

3.3. Redox-Dependent Antioxidant Activity and Selective Cytotoxicity of Ceria NPs

Ceria NPs have attracted considerable attention due to their unique redox properties, which arise from the coexistence of Ce³⁺ and Ce⁴⁺ oxidation states on their surface. This redox flexibility enables CeO₂ NPs to act as either antioxidants or pro-oxidants depending on the surrounding microenvironment, making them particularly interesting for biomedical applications. In this study, the redox activity of Ceria NPs was first evaluated using the DPPH radical scavenging assay. Ceria NPs demonstrated strong, dose-dependent DPPH scavenging activity, reaching 72.01 ± 2.76% inhibition at 0.5 mg mL^–1 (Figure a,b). The relatively high scavenging efficiency observed in the DPPH assay suggests that the redox-active surface of Ceria NPs, particularly the reversible Ce³⁺/Ce⁴⁺ cycling and oxygen vacancies, effectively facilitates electron transfer reactions with DPPH radicals. The scavenging activity of the standard antioxidant Trolox was used as a positive control, yielding an IC₅₀ value of 0.46 ± 0.02 μg mL^–1 (Supporting Information Figure S2), thereby confirming the reliability and sensitivity of the experimental system.

2.

Antioxidant activity and cytotoxicity of Ceria NPs. (a) DPPH radical scavenging activity. (b) Bar graph representing the percentage of DPPH radical scavenging activity. Statistical analysis was performed using one-way ANOVA. (c) Cytotoxicity of Ceria NPs on MCF-10A, MCF-7, and MDA-MB-231. 1% TX was use as a positive control. Statistical significance was determined using two-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001, and ****p < 0.0001 vs control. All data are presented as mean ± SEM (n = 3).

To further explore the biological implications of this redox behavior, cell viability was assessed using the MTT assay in both a nonmalignant human breast epithelial cell line (MCF-10A) and human breast cancer cell lines (MCF-7, and MDA-MB-23). 1% Triton-X (TX) was used as a positive control in this assay. As shown in Figure c, Ceria NPs exerted minimal cytotoxic effects on normal breast cells (MCF-10A), with cell viability remaining high across all tested concentrations. This observation is consistent with their antioxidant properties, which may help protect normal cells from oxidative damage. In contrast, Ceria NPs exhibited dose-dependent cytotoxicity toward breast cancer cells, with an IC₅₀ of 363.0 ± 71.89 μg mL^–1 in MCF-7 cells and more pronounced reduction in viability in the aggressive MDA-MB-231 cell line (325.9 ± 16.71 μg mL^–1). Notably, the higher sensitivity of MDA-MB-231 cells may be account to their elevated basal ROS levels and compromised antioxidant defense systems, which render TNBC cells more susceptible to redox perturbations.

The observed differential response between normal and cancer cells may be attributed to the environment-dependent redox activity of CeO₂ NPs. Under physiological conditions (neutral pH), CeO₂ NPs predominantly function as antioxidants, as supported by the DPPH assay results, thereby contributing to their biocompatibility in normal cells. However, in the tumor microenvironmentcharacterized by acidic pH, altered redox balance, and elevated levels of reactive oxygen species (ROS)CeO₂ NPs are known to undergo a functional shift toward pro-oxidant behavior. This shift can enhance intracellular ROS generation, leading to oxidative stress, mitochondrial dysfunction, and ultimately cancer cell death. Furthermore, cancer cells are generally more susceptible to oxidative stress due to their already elevated basal ROS levels. Therefore, the additional ROS induced by CeO₂ NPs may exceed the cellular antioxidant capacity, triggering apoptosis or other forms of cell death. In contrast, normal cells with lower basal ROS levels are better equipped to maintain redox homeostasis, which may explain their resistance to CeO₂ NP-induced toxicity.

Taken together, these findings suggest that CeO₂ NPs possess a dual redox function, acting as antioxidants under normal physiological conditions while exhibiting pro-oxidant and cytotoxic effects in cancerous environments. This selective behavior highlights their potential as a promising nanomaterial for targeted cancer therapy.

3.4. Drug Loading, Characterization, and In Vitro Release Study of Ceria-Dox

Both previous studies and the present findings confirm that Ceria NPs exhibiting low toxicity in normal cells and redox-selective behavior represent a promising drug delivery platform. However, despite their selective cytotoxicity, the relatively high IC₅₀ values observed for CeO₂ NPs alone indicate that further enhancement of anticancer efficacy is required. Doxorubicin (Dox), a widely used chemotherapeutic agent, has, however, its clinical application limited by severe side effects. Nanocarrier-based delivery has emerged as an effective strategy to mitigate these effects by improving drug selectivity and controlled release. Therefore, loading Dox onto CeO₂ NPs may enhance cytotoxic effects in breast cancer cells while potentially reducing systemic toxicity.

In this study, we successfully loaded dox on to Ceria NPs. As shown in Figure a, both Dox and the supernatant collected after loading (unbound Dox) exhibited a characteristic UV–vis absorption peak at 495 nm, while bare Ceria NPs showed a distinct peak at 290 nm (Supporting Information Figure S3), consistent with previous reports. These results, together with the fluorescence spectrum of Dox-loaded CeO₂ NPs (Figure b), confirm successful conjugation. Free Dox displayed a single emission peak at 584 nm, within its typical fluorescence range (560–590 nm). Whereas Ceria-Dox displayed dual emissions at 555 and 584 nm, indicating a slight spectral shift upon interaction with the nanoparticle surface. Drug loading efficiency (LE) was determined by analyzing the UV–vis absorbance spectra of the supernatants after loading and calculating the difference between the initial and remaining Dox concentrations, yielding ∼80% loading. This value surpasses many conventional nanocarriers, such as liposomes (typically 50–60%), polymeric nanoparticles (55–70%), and AuNP-Dox systems (72–74%). This high loading efficiency can be attributed to the unique properties of our Ceria NPs, including a high surface area, , the presence of surface hydroxyl groups and oxygen vacancies that enable strong electrostatic and hydrogen-bonding interactions with Dox, , and excellent colloidal stability in biological media. In addition, Fourier transform infrared (FTIR) spectroscopy and DLS analyses of Ceria-Dox were performed and are provided in the Supporting Information to further confirm successful drug loading and evaluate nanoparticle behavior in biologically relevant environments (Supporting Information Figure S4, Supporting Information Table S1).

3.

Optical and fluorescence properties of Ceria NPs and Ceria-Dox. (a) UV–vis spectra, and (b) fluorescence emission spectra. (c) In vitro cumulative release profile of free DOX (red) and Dox from Ceria-Dox (blue) was evaluated in PBS solution. Data are expressed as mean and nonlinear regression was applied for curve fitting. (d) Fluorescence image of bare Ceria (left) and Ceria-Dox (right) under UV- light.

The release profiles of Dox from Ceria NPs was further investigated in 1× PBS at 0, 0.5, 1, 2, 4, 6, 24, 48, and 72 h. Evaluating the drug release profile is important to understand release kinetics and ensure controlled, sustained delivery for improved efficacy and reduced toxicity. As shown in Figure c, Dox release from Ceria NPs is shown as a biphasic pattern or drug release in two distinct stages. An initial burst release of approximately ∼31.4% was observed within the first 24 h, followed by a plateau phase, where only a slight increase was observed at 48 h (∼35.08%) and 72 h (∼35.33%). This modest initial burst, followed by a plateau, suggests strong electrostatic interactions between Dox and the Ceria NP surface, resulting in diffusion-limited release. The release profile is consistent with previous studies that showed slower release at physiological pH and accelerated release in acidic tumor microenvironment in micelles, Au-NPs, and CeO₂/Dox combination with glutathione (GSH) loaded with Dox. These results indicate that Ceria NPs provide a controlled and sustained release of Dox, which could enhance tumor-targeted delivery while minimizing systemic exposure. Drug loading was further visuallized under UV illumination (Figure d). The bare Ceria-NPs suspension displayed negligible fluorescence (left), whereas the Ceria–Dox suspension emitted distinct fluorescence (right), indicating successful Dox incorporation onto the CeO₂ particles.

3.5. In Vitro Release Kinetics Study of Ceria-Dox

Following the experimental release study, mathematical modeling was used to evaluate the release kinetics of Dox from cerium oxide nanoparticles, which are known to improve therapeutic efficacy, minimize systemic toxicity, and enable controlled drug release. ,

This study evaluated the application of three distinct models, namely Korsmeyer-Peppas, Peppas-Sahlin, and Weibull, to analyze Dox dissolution data. The analysis was performed using DDSolver, an add-in program for Microsoft Excel, and the curve fitting profiles are presented in Figure . The mean values of the fitted parameters from three independent experiments are summarized in Table . The release profiles of Dox were fitted to the three kinetic models, all of which exhibited strong concordance with the experimental data (R ² > 0.95), indicating that each model provides a reasonable approximation of Dox release from Ceria NPs. Comparison of R ² values identified the Peppas–Sahlin model as the best descriptor of the release kinetics, suggesting that drug release is governed by a combination of Fickian diffusion and polymer relaxation.

4.

Kinetic analysis of Dox release from Ceria-Dox. Experimental cumulative release data from three independent experiment were fitted to the Korsmeyer–Peppas (a–c), Peppas–Sahlin (d–f), and Weibull (g–i) models, illustrating the agreement between observed and predicted release profiles.

2. Fitted Parameters for the Korsmeyer–Peppas, Peppas–Sahlin, and Weibull Models, Detailing the Release Kinetics of Dox from Ceria NPs.

models	equations	parameters	R 2
Korsmeyer–Peppas	F = kt n	k = 8.220	0.959
		n = 0.365
Peppas–Sahlin	F = k 1 t m  + k 2 t 2m	k 1 = 6.610	0.998
		k 2 = −0.301
		m = 0.601
Weibull	F = 100 $(1-e\frac{{(t-Ti)}^{\beta }}{\alpha })$	α = 10.264	0.979
		β = 0.376
		Ti = 0.464

It should be noted that the kinetic constant k ₂ in the Peppas–Sahlin model cannot assume a negative value from a physical standpoint, as this would imply an unphysical negative contribution of polymer relaxation to overall drug release. Observed negative values typically indicate either that the model is not fully representative of the system or that polymer relaxation contributes negligibly to the release mechanism. Analysis using the Korsmeyer–Peppas model yielded a diffusional exponent n = 0.365, indicating predominantly Fickian diffusion. The Weibull model further characterized the release kinetics, with a shape parameter β = 0.376 (<1) consistent with a biphasic release profile, comprising an initial burst followed by a slower, sustained phase. The lag time parameter Ti was determined to be 0.464 h (∼28 min), in agreement with literature reports where Ti is typically near zero.

3.6. Cytotoxicity of Doxorubicin-Loaded Cerium Oxide Nanoparticles (Ceria-Dox)

Since bare CeO₂ nanoparticles exhibited relatively high IC₅₀ values in cancer cells, consistent with previous reports in ovarian carcinoma cell lines (A2780, C200, and SKOV3), highlighting their limited standalone cytotoxicity, further enhancement strategies are required. Doxorubicin (Dox), although highly effective against cancer cells, is associated with significant side effects, including cardiotoxicity and damage to normal tissues. Consistent with this, our results (Figure a) demonstrate that the effective dose of Dox required to induce cytotoxicity in TNBC cells (MDA-MB-231) also reduces the viability of normal cells (MCF-10A), indicating a lack of selectivity. Therefore, combining Dox with CeO₂ nanoparticles may provide a strategy to enhance anticancer efficacy while minimizing off-target toxicity.

5.

Cell viability after treatment with Dox and Ceria-Dox. Cytotoxicity of bare Doxorubicin (a), and Ceria-Dox (b) on MCF-10A, MCF-7, and MDA-MB-231. All data are presented as mean ± SEM (n = 3). 1% Triton-X (TX) was use as a positive control. Statistical significance was determined using two-way ANOVA. ****p < 0.0001 vs control. All data are presented as mean ± SEM (n = 3).

To overcome this, Dox was loaded onto CeO₂ NPs to potentiate anticancer activity and reduce side effects. Treatment with Ceria–Dox (Figure b) demonstrated a more selective cytotoxic profile. While cell viability decreased in all cell lines with increasing nanoparticle concentration, the reduction was more pronounced in cancer cells compared to MCF-10A cells. At higher concentrations (200–400 μg mL^–1), Ceria–Dox significantly reduced the viability of MCF-7 and MDA-MB-231 cells, whereas MCF-10A cells retained relatively higher viability. Statistical analysis indicated significant differences between normal and cancer cells at these concentrations. Furthermore, MDA-MB-231 cells exhibited greater sensitivity to Ceria–Dox treatment compared to MCF-7 cells, as reflected by the lower cell viability at equivalent concentrations. This differential response may be attributed to the distinct biological characteristics of the two breast cancer subtypes. MCF-7 cells are hormone receptor-positive (ER-positive) breast cancer cells with relatively lower basal oxidative stress, whereas MDA-MB-231 cells represent an aggressive TNBC subtype characterized by enhanced invasiveness, elevated metabolic activity, and increased dependence on redox homeostasis. Due to their intrinsically high ROS levels, TNBC cells are particularly vulnerable to additional oxidative imbalance and ROS-mediated damage. Therefore, the combined effects of Dox-induced ROS generation and the redox-modulating properties of Ceria NPs may preferentially increase oxidative stress in MDA-MB-231 cells, resulting in greater cytotoxicity than in MCF-7 cells. This enhanced efficacy suggests a synergistic effect between the chemotherapeutic action of Dox and the redox-modulating properties of Ceria NPs (Table ). Specifically, while Dox induces cytotoxicity primarily through DNA intercalation and ROS generation, Ceria NPs may further amplify oxidative stress in cancer cells, particularly in TNBC subtypes that are highly sensitive to ROS imbalance. Notably, a reduction in cell viability after treat MCF-10A cells with Ceria-Dox is likely associated with the intrinsic toxicity of Dox and its partial release from the nanoparticle surface. However, Ceria–Dox did not achieve 50% growth inhibition at the highest tested concentration, precluding IC₅₀ determination and indicating low toxicity toward normal cells, suggesting that nanoparticle-mediated delivery may reduce off-target toxicity while maintaining anticancer efficacy. Similar findings have been reported in previous studies, where nanocarrier systems improved the therapeutic index of Dox by enhancing tumor-specific cytotoxicity while minimizing damage to normal tissues. ,

3. IC₅₀ Values (μg mL^–1) of Ceria NPs, Ceria–Dox, and Dox in Cancer and Normal Cell Lines,

IC50	MCF-7 (μg mL–1)	MDA-MB-231 (μg mL–1)	MCF-10A (μg mL–1)
Ceria NPs	363.0 ± 71.89	325.90 ± 16.71	N/A
Ceria-Dox	142.80 ± 18.46	60.91 ± 7.67	N/A
free Dox	0.69 ± 0.07	1.96 ± 0.33	2.74 ± 0.52

^aValues are mean ± SEM (n = 3).

^bN/A = non applicable.

Overall, these results demonstrate that Ceria–Dox not only enhances anticancer efficacy, particularly against aggressive TNBC cells, but also offers a potential strategy to mitigate the systemic toxicity associated with free Dox, thereby supporting its application as a promising nanotherapeutic platform for breast cancer treatment. Given the heightened sensitivity of TNBC cells, the anticancer efficacy of Ceria–Dox was further evaluated in MDA-MB-231 cells.

3.7. Live/Dead Cells Staining Elucidates the Anticancer Effect of Ceria-Dox-NPs

To further confirm the cytotoxic effect of Ceria-Dox on TNBC, the live/dead assay was conducted using calcein-AM and propidium iodide (PI) staining. Calcein-AM is converted by intracellular esterases into calcein, which is retained in the cytoplasm of viable cells and emits green fluorescence, whereas PI selectively penetrates cells with compromised membranes and stains the nuclei red, indicating cell death. The live/dead analysis revealed a reduction in calcein-derived green fluorescence alongside an increase in PI-stained cells with higher concentrations of Ceria-Dox, indicating dose-dependent cytotoxicity (Figure ). Our results confirmed that NPs induced the cytotoxic effect in a significant, dose-dependent manner in MDA-MB-231 cells.

6.

Live/dead cells assay of MDA-MB-231 cells after Ceria-Dox treatment. Calcein-AM is stained for viable cells (green fluorescence), while propidium iodide (PI) stains dead cells. Scale bar = 20 μm.

3.8. Mechanistic Investigation of Ceria Dox in Breast Cancer Cells

To further elucidate the molecular mechanisms underlying the enhanced anticancer activity of Ceria-Dox, intracellular ROS generation and mitochondrial membrane potential (MMP) disruption were investigated using DCFDA and JC-1 staining assays, respectively (Figure ). Oxidative stress and mitochondrial dysfunction are widely recognized as crucial mechanisms underlying nanoparticle-mediated cancer cell death and apoptosis. In particular, excessive ROS accumulation can disrupt cellular redox homeostasis, leading to oxidative damage of biomolecules and activation of mitochondria-dependent apoptotic pathways. Since cerium oxide nanoparticles possess redox-active properties associated with the reversible transition between Ce³⁺ and Ce⁴⁺ oxidation states, evaluating ROS-related mechanisms is important for understanding the therapeutic activity of the Ceria–Dox system.

7.

Intracellular ROS production and MMP disruption induced by Ceria-Dox. (a) Intracellular ROS generation. Representative fluorescence images (left) and quantitative analysis by microplate reader (right) of DCFDA. Scale bar = 100 μm. (b) JC-1 staining images (left) showing JC-1 monomers (green) and aggregates (red), with merged images. Scale bar = 20 μm. Quantification by microplate reader (right) shows the red/green fluorescence intensity ratio normalized to control. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01 vs control.

As shown in Figure a, DCFDA staining revealed increased intracellular ROS generation following Ceria Dox treatment. Compared with untreated control cells, cells treated with 12.5 and 25 μg/mL Ceria Dox exhibited markedly enhanced green fluorescence intensity, indicating elevated ROS accumulation within the cells. The observed elevation in ROS may result from combined effects of doxorubicine-induced ROS generation and the redox activity of Ceria NPs, which can exhibit pro-oxidant behavior. Together, these effects likely enhance oxidative stress and contribute to improved anticancer activity.

To further investigate whether ROS overproduction affected mitochondrial function, MMP was subsequently evaluated using JC-1 staining (Figure b). Untreated cells showed strong red fluorescence, indicating intact MMP, whereas Ceria-Dox-treated cells exhibited a concentration-dependent shift from red to green fluorescence, consistent with mitochondrial depolarization. Quantitative analysis of the red/green fluorescence ratio confirmed a significant MMP reduction after treatment. Since mitochondria depolarization is an early hallmark of mitochondria-mediated apoptosis, these findings suggest activation of apoptotic signaling pathways.

Notably, the concurrent increase in ROS generation and loss of MMP indicates that oxidative stress plays a central role in the cytotoxic mechanism of Ceria Dox. The sustained ROS accumulation induced by Ceria Dox likely overwhelms cellular antioxidant defense, resulting in irreversible mitochondrial damage and apoptosis. Furthermore, the nanoparticle-based delivery system may enhance intracellular Dox retention, thereby intensifying oxidative damage. Collectively, these findings suggest that the enhanced anticancer activity of Ceria Dox is driven by ROS-mitochondrial apoptosis, supporting its potential as an effective nanoparticle-based cancer therapy platform.

3.9. Ceria–Dox Suppresses Clonogenic Growth of Breast Cancer Cells

ROS and MMP assays represent early intracellular responses to Ceria–Dox treatment and are mechanistically linked to downstream functional outcomes, particularly the long-term survival and proliferative capacity of cancer cells. Persistent oxidative stress and mitochondrial dysfunction are known to impair DNA integrity, cellular repair mechanisms, and clonogenic potential, ultimately determining whether cancer cells can sustain indefinite proliferation following treatment. Therefore, the effect of Ceria–Dox on the clonogenic survival of breast cancer cells was further evaluated using a colony formation assay.

The colony formation assay was performed following treatment with increasing concentrations of Ceria–Dox (10, 30, and 50 μg mL^–1). As shown in Figure a, Ceria–Dox inhibited colony formation in a dose-dependent manner in MDA-MB-231 compared with untreated controls. A decrease in colony number was observed with increasing nanoparticle concentrations, with the strongest inhibitory effect detected at 50 μg mL^–1 (Figure b). These findings indicate that Ceria–Dox exerts not only acute cytotoxic effects but also prolonged inhibitory effects on cancer cell reproductive survival.

8.

Effect of Ceria-Dox on colony formation of MDA-MB-231 cells. (a) Representative images of colony formation in MDA-MB-231 cells treated with Ceria-Dox at concentrations of 0, 10, 30, and 50 μg mL^–1 for 7 days (b) quantitative analysis of colony formation expressed as percentage relative to the untreated control. Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined compared with the control group (*p < 0.05, ****p < 0.0001).

Overall, these results demonstrate that Ceria–Dox effectively suppresses the long-term tumorigenic and proliferative potential of breast cancer cells. The ability of Ceria–Dox to inhibit colony formation further supports its potential as a multifunctional nanotherapeutic platform that targets multiple hallmarks of cancer progression, rather than relying solely on short-term cytotoxicity.

3.10. Inhibition of Cancer Cell Migration and Invasion by Ceria-Dox

Metastasis is a hallmark of TNBC and is strongly associated with poor prognosis. Elevated intracellular ROS levels in TNBC cells promotes migration and invasion by activating redox-sensitive signaling pathways involved in epithelial–mesenchymal transition (EMT) and matrix degradation. Given the redox-modulating capability of Ceria NPs in cancer cells, Ceria–Dox may disrupt these processes and inhibit metastatic progression. Accordingly, transwell migration and invasion assays were performed. As shown in Figure a, Ceria–Dox markedly suppressed MDA-MB-231 cell migration and invasion compared to untreated controls. Quantitative analysis (Figure b) demonstrated a significant, dose-dependent inhibition of cell migration. Similarly, invasion assays (Figure c) showed a concentration-dependent decrease in invasive capacity. These findings indicate that Ceria–Dox not only induces cytotoxicity but also suppresses the migratory and invasive capacity of TNBC cells. This effect is likely mediated by ROS modulation via Ce³⁺/Ce⁴⁺ redox cycling and oxygen vacancies, which can disrupt cytoskeletal dynamics, impair epithelial–mesenchymal transition (EMT), and reduce motility-associated signaling. In addition, apoptosis induction may further contribute to the loss of cell migration and invasion.

9.

Effects of Ceria–Dox on migration and invasion of MDA-MB-231 cells. (a) Representative images of transwell migration and invasion assay. Quantitative analysis of transwell migration (b) and invasion assay (c). Data are presented as mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA, where *p < 0.05, **p < 0.01, and ***p < 0.001 vs control.

Collectively, the dual functionality of Ceria–Dox in promoting cancer cell death while inhibiting metastatic behavior highlights its potential as a therapeutic strategy for TNBC. Further in vivo studies are required to validate its efficacy and safety, particularly in the context of tumor microenvironment complexity, systemic distribution, and drug release behavior.

4. Conclusion

Ceria NPs were successfully synthesized via a hydrothermal method and exhibited excellent colloidal stability in serum-containing medium (10% FBS) with a hydrodynamic diameter of 56.3 nm. Their physicochemical properties were largely preserved after Dox loading, resulting in stable nanocomplexes with physicochemical characteristics suitable for efficient cellular delivery. The Ceria-Dox system exhibited controlled drug release and markedly enhanced cytotoxicity toward both MCF-7 cells and highly aggressive triple-negative MDA-MB-231 breast cancer cells. Beyond drug delivery, the multifunctional nature of Ceria NPs also imparts antimigratory and anti-invasive effects. However, this study is limited to in vitro evaluations, which do not fully capture in vivo complexity such as pharmacokinetics, biodistribution, immune responses, and systemic toxicity. Therefore, in vivo studies are necessary to further validate efficacy, safety, and translational potential. Overall, these findings highlight Ceria-Dox as a promising nanoplatform for selective and multifunctional breast cancer therapy, warranting further mechanistic investigation, optimization, and evaluation of tumor models.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c03856.

• Colloidal stability of nanoparticles (Figure S1); Antioxidant activity of trolox (Figure S2); Absorbance of Ceria NPs (Figure S3); FTIR spectra (Figure S4); Size, polydispersity index (PdI), and zeta potential of Ceria Dox (Table S1) (PDF)

A.W.: methodology, validation, formal analysis, investigation, data curation, visualization, and writingoriginal draft. S.K.: methodology, validation, formal analysis, investigation, data curation, visualization, and writingreview. W.S.: methodology, validation, formal analysis, and writingreview. P.L.: methodology, validation, and formal analysis, writingreview, and funding acquisition. D.B.: methodology, validation, formal analysis, writingreview, and funding acquisition. K.P.K.: conceptualization, methodology, resources, writingreview and editing, supervision, project administration, and funding acquisition.

The authors declare no competing financial interest.