# Study of the effect of ultrasonication on MnO2: Morphological evolution from nanosheets to nanofibers and its implications for biomedical redox modulation

Mahran Bechetnia, Oumaima Hajlaoui, Julien Boudon, Michaële Herbst, R.

Chassagnon, Michel Meyer, Nadine Millot, Latifa Bergaoui

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## ► To cite this version:

Mahran Bechetnia, Oumaima Hajlaoui, Julien Boudon, Michaële Herbst, R. Chassagnon, et al.. Study of the effect of ultrasonication on MnO2: Morphological evolution from nanosheets to nanofibers and its implications for biomedical redox modulation. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2026, 735, pp.139481. 10.1016/j.col SURFA.2026.139481. hal-05452278

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# Study of the effect of ultrasonication on MnO₂: Morphological evolution from nanosheets to nanofibers and its implications for biomedical redox modulation

Mahran Bechetnia<sup>a,b</sup>, Oumaima Hajlaoui<sup>a</sup>, Julien Boudon<sup>b</sup>, Michaële Herbst<sup>b</sup>, Rémi Chassagnon<sup>b</sup>, Michel Meyer<sup>c</sup>, Nadine Millot<sup>b,*</sup>, Latifa Bergaoui<sup>a,*</sup>

## HIGHLIGHTS

* Ultrasonication transforms birnessite from stacked nanosheets into nanofibers.

* Crystallite size drops to ~3.1 nm and microstrains rate rises, indicating more defects.

* Nanofibers deplete glutathione faster, enhancing redox modulation.

* Nanosheets generate slightly more OH\* from H₂O₂ due to higher surface Mn³⁺ content.

## GRAPHICAL ABSTRACT

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

**Keywords:**

Triclinic birnessite

Ultrasonication

Morphology-dependent reactivity

Glutathione (GSH) depletion

Reactive oxygen species (ROS) generation

## ABSTRACT

Triclinic birnessite (TMA-birnessite) was synthesized and systematically transformed through controlled ultra-sonication (10–30 min) to optimize its morphology and redox reactivity for biomedical use. Transmission electron microscopy revealed a progressive morphological evolution: from stacked nanosheets to individual monolayers, and ultimately to elongated nanofibers, driven by anisotropic fragmentation along Mn(III)O₆ chains. X-ray diffraction analysis (Williamson-Hall model) indicated a 70 % reduction in crystallite size (from 9.6 to 3.1 nm) and a sixfold increase in microstrains (from 0.2 % to 1.3 %), reflecting extensive defect generation. Ultrasonication enhanced colloidal stability in aqueous media, as demonstrated by improved zeta potential (from –10 mV to –35 mV) and reduced hydrodynamic diameter (from 544 nm to 173 nm), though stability remained limited under physiological ionic strength. Kinetic studies using a pseudo-first-order model revealed morphology-dependent redox reactivity toward glutathione (GSH). Nanofibers exhibited a 1.5-fold faster GSH-mediated reduction rate (k ≈ 0.097 vs. 0.061 min⁻¹ for nanosheets at 4.0 mM GSH), indicating superior antioxidant depletion—critical for targeting tumor microenvironments. Conversely, methylene blue assays indicated a higher oxidative activity for nanosheets, likely related to their higher Mn(III) surface content (41 vs. 33 % in nanofibers), but this test alone does not allow unambiguous identification of the reactive species involved. This

<footnote><sup>a</sup> University of Carthage, National Institute of Applied Sciences and Technology, EcoChimie Laboratory, Tunis, Tunisia</footnote>

<footnote><sup>b</sup> Université Bourgogne Europe, CNRS, Laboratoire Interdisciplinaire Carnot de Bourgogne ICB UMR 6303, 21000 Dijon, France</footnote>

<footnote><sup>c</sup> Université Bourgogne Europe, CNRS, Institut de Chimie Moléculaire de l'Université de Bourgogne ICMUB UMR 6302, 21000 Dijon, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: nadine.millot@ube.fr (N. Millot), latifa.bergaoui@insat.ucar.tn (L. Bergaoui).</footnote>
interpretation was therefore validated using the terephthalic acid fluorescence probe, which specifically confirmed hydroxyl radical generation in the MnO₂/H₂O₂ system and revealed a stronger OH⁺ production for nanosheets than for nanofibers, particularly in the presence of GSH. These results establish a clear morphology-activity relationship: nanofibers primarily act as GSH scavengers, while nanosheets serve as ROS generators. This tunability positions ultrasonicated birnessite as a promising platform for biomedical applications requiring balanced antioxidant depletion and ROS generation.

# 1. Introduction

The rapid progress in nanotechnology has profoundly impacted biomedical research, particularly through the development of metal oxide nanomaterials with tunable physicochemical properties. Among these, manganese dioxide (MnO₂) has attracted significant attention due to its versatile redox chemistry and structural diversity [1,2]. Notably, δ-MnO₂ (birnessite) features a layered structure composed of edge-sharing MnO₆ octahedral sheets, with hydrated cations and water molecules occupying the interlayer spaces [3].

This lamellar arrangement typically yields anisotropic, platelet-like aggregates composed of stacked nanosheets (Ns) [3,4]. The term “tactoid” (Fig. 1a) is sometimes used by analogy to describe such assemblies, however, its use for solid-state MnO₂ remains debated and is more commonly associated with liquid-crystalline phases [3]. These stacked nanosheets offer high surface area, tunable porosity, and redox-active interlayers, making them promising for biomedical applications such as drug delivery, biosensing, and bioimaging [4,5]. Recent evidence suggests that anisotropic morphologies can improve retention and localized accumulation of therapeutic agents in targeted tissues, including tumors [5].

The triclinic birnessite consists of layers formed by alternating chains of Mn(III)O₆ and Mn(IV)O₆ octahedra (Fig. 1b). The layers carry a permanent negative charge due to the partial substitution of Mn(IV) by Mn(III), which is maintained across a wide pH range [6].

δ-MnO₂ Ns have emerged as promising platforms for a range of biomedical applications, particularly due to their involvement in essential redox processes that underpin many physiological functions [3,7]. One key redox interaction is the reaction of MnO₂ with glutathione (GSH), a major intracellular antioxidant. In cancer cells, elevated GSH levels protect them against oxidative damage; MnO₂ can oxidize GSH to glutathione disulfide (GSSG), thereby depleting intracellular GSH and sensitizing cancer cells to ROS-based therapies [3,8]. Moreover, GSH-mediated reduction of MnO₂ to Mn²⁺ in the tumor microenvironment can trigger dissolution of manganese oxide carriers, enabling drug release preferentially within tumor cells while limiting exposure to healthy tissues [3]. Mn²⁺ ions also serve as effective MRI contrast agents, enhancing bioimaging capabilities [9]. Recent work by Wen et al. further demonstrates the potential of MnO₂ to improve treatment outcomes by exploiting its adsorption and catalytic properties [10]. When combined with photothermal and photodynamic therapies, MnO₂ nanosystems amplify therapeutic efficacy against tumors while reducing off-target effects [11]. In summary, MnO₂ nanoparticles hold significant promise for cancer treatment by modulating the tumor microenvironment, enhancing drug delivery and imaging, and enabling integrated

diagnostic and therapeutic strategies.

As previously noted, birnessite exhibits a stacked-layer structure, forming micrometer-sized tactoids. However, many biomedical applications require nanomaterials with high surface area and dispersibility, necessitating the delamination of these tactoids into individual Ns. Tang et al. demonstrated that ultrasonication effectively facilitates this delamination, thereby enhancing dispersion in biological solvents [12]. Beyond their reactivity with glutathione, MnO₂ nanostructures can catalyze the generation of reactive oxygen species (ROS), such as hydroxyl radicals, in the presence of hydrogen peroxide. This process can be monitored using methylene blue (MB) as a ROS probe, offering valuable insights into their potential as oxidative stress amplifiers in cancer therapy [13].

While ultrasonication has been widely used to delaminate layered materials, its effects on the morphological, structural, and redox properties of triclinic birnessite—especially in biomedical contexts—remain poorly characterized. This study bridges this knowledge gap by systematically investigating the impact of ultrasonication duration on morphological evolution, defect generation, and redox reactivity toward both GSH and ROS.

# 2. Experimental section

## 2.1. Chemicals

All reagents were purchased from Fisher Scientific and used as received, unless otherwise specified. The following chemicals were used: manganese(II) chloride tetrahydrate (MnCl₂·4 H₂O; ACS reagent, ≥ 99 %), tetramethylammonium hydroxide (TMAOH; 25 % w/w in H₂O), hydrogen peroxide (H₂O₂; 30 % w/w), reduced L-glutathione (GSH; ≥ 97 %), ammonium iron(II) sulfate hexahydrate (Mohr's salt; ≥ 98 %), potassium permanganate (KMnO₄; ≥ 97 %), sodium pyrophosphate dibasic (Na₂H₂P₂O₇; ≥ 97 %), and hexaamminecobalt(III) chloride ([Co(NH₃)₆]Cl₃; ≥ 99 %). For Mn average oxidation state (AOS) determination, Mohr's salt, KMnO₄, Na₂H₂P₂O₂, and Co(NH₃)₆Cl₃ were employed. Phosphate-buffered saline (PBS, 1 ×) and sodium chloride (NaCl; 0.15 M) were used to maintain physiological ionic strength. The MB assay utilized MB (Basic Blue 9; ≥ 82 %) and sodium bicarbonate (NaHCO₃; ≥ 99.5 %). Terephthalic acid (TA; ≥ 98 %) was used as a fluorescence probe for detecting hydroxyl radicals. Ultrapure water (18.2 MΩ cm; Veolia system) was used in all experiments.

## 2.2. Synthesis of tetramethylammonium-birnessite

Tetramethylammonium-birnessite was synthesized following established literature procedures [14–18]. 12 mL of 1 M TMAOH aqueous solution and 2 mL of 30 % (w/w) H₂O₂ were diluted to a final volume of 20 mL with ultrapure water. This solution was then rapidly added to 10 mL of 0.3 M MnCl₂·4 H₂O, resulting in an immediate color change due to the oxidation of Mn(II) to Mn(IV). The mixture was stirred vigorously at room temperature (~25 °C) for 12 h.

The resulting suspension was centrifuged at 9000 g for 10 min and washed repeatedly with ultrapure water and ethanol until the absence of chloride ions was confirmed by the AgNO₃ test. The final product (TMA-birnessite) was dried in an oven at 60 °C for 24 h.

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Schematic representation of (a) the tactoid of a lamellar structure and (b) two stacked layers of triclinic birnessite with an interlayer.</figcaption></figure>
## 2.3. Ultrasonication conditions

TMA-birnessite (10 mg) was dispersed in 20 mL of ultrapure water and ultrasonicated for 10, 20, or 30 min using a VCX-750 ultrasonic probe (750 W, 60 % amplitude). Sonication was applied in pulsed mode (2 s on, 3 s off), with the suspension maintained below 27 °C in an ice bath to prevent overheating; a critical adjustment to preserve structural integrity during fragmentation.

After treatment, suspensions were centrifuged at 9 000 g for 10 min. This process sedimented tactoids, while delaminated monolayers remained in the supernatant, which was collected and stored at 4 °C. Water was removed by freeze-drying (FreeZone 4.5 L, -84 °C collector, 230 V-50 Hz, Labconco, Kansas City, MO, USA; stainless-steel chamber, cat. 71040).

## 2.4. Characterization techniques

X-ray diffraction (XRD) patterns were collected using a Bruker D8 0-θ diffractometer equipped with a Cu Kα radiation source (λ = 1.5406 Å), operated at 40 kV and 40 mA. Data acquisition covered a 2θ range of 5° to 100°, with a step size of 0.0307° and a counting time of 960 s per step. To improve particle statistics and minimize preferred orientation effects, the sample was continuously rotated at 5 rpm during acquisition.

Instrumental broadening was determined using the NIST SRM 1976b standard reference material (a sintered alumina disc certified for peak positions and relative intensities). The full width at half maximum (FWHM) for each reflection, βm, was obtained by fitting diffraction peaks with a Lorentzian function. The corrected FWHM, βc, was then calculated using Eq. 1:

$$ \beta_c^2 = \beta_m^2 - \beta_f^2 \quad (1) $$

where βi is the instrumental broadening extracted from the SRM 1976b reference. No Kα2 stripping was applied; both Kα1 and Kα2 contributions were retained during peak analysis.

Crystallite size (D) and microstrain rate (ε) were determined using the Williamson-Hall (W-H) method, according to Eq. 2:

$$ \beta \cos(\theta) = \frac{K \lambda}{D} + 4 \epsilon \sin(\theta) \quad (2) $$

where K is the shape factor, set to 0.9. A linear regression of β cos(θ) versus sin(θ) was performed, with the intercept (Kλ/D) used to calculate crystallite size and the slope (4ε) to determine microstrain rate.

Morphological characterization was performed using Transmission Electron Microscopy (TEM). Conventional TEM images were acquired with a Hitachi HT7800 microscope operated at 100 kV (0.36 nm resolution), while high-resolution TEM (HRTEM) observations were conducted using a JEOL JEM-2100F microscope at 200 kV. Samples were prepared by drop-casting a diluted aqueous suspension of MnO₂ onto carbon-coated copper grids, followed by solvent evaporation.

Particle size distributions were determined by Dynamic Light Scattering (DLS) using a Malvern Nano ZS instrument equipped with DTS Nano V7.11 software. Zeta potential measurements were performed with a Malvern Zetasizer Nano ZSP. Suspensions were prepared in 10⁻² M NaCl aqueous solutions, and their pH was adjusted from 2 to 10 using an autotitrator with NaOH (0.1 and 0.01 M) or HCl (0.1 and 0.01 M) solutions.

For each condition, three independent measurements were carried out on freshly prepared dispersions (n = 3). The reported values correspond to the mean ± standard deviation; the error bars shown in the figures represent this standard deviation.

Raman spectra were obtained using a Renishaw inVia microspectrometer utilizing a 532 nm laser excitation and a 50 × microscope objective.

Specific surface area, pore volume, and pore size distribution were determined using the BET and BJH methods, based on N₂ adsorption-desorption isotherms measured at 77 K with a Micromeritics TriStar II

apparatus. BET surface areas are reported with an instrumental uncertainty lower than 4 %, according to the manufacturer's specifications.

X-ray Photoelectron Spectroscopy (XPS) analysis was performed using a PHI 5000 VersaProbe instrument with monochromatic Al Kα radiation (E=Kα(Al) = 1486.7 eV, spot size 200 µm). Suspensions were deposited onto a silicon substrate and dried under vacuum in a desiccator. After Shirley background subtraction, spectra were fitted using non-linear least squares regression in CasaXPS software. To compensate for charging effects, a neutralization method was applied and the C1s peak at 284.8 eV was used as an internal reference for energy calibration. Peak shapes were modeled using a Voigt function, combining Gaussian and Lorentzian components. For improved accuracy, the Lorentzian full width at half maximum was constrained according to the core-hole lifetime, while the Gaussian component was adjusted to account for instrumental and charge-induced broadening.

Nitrogen content was quantified by combustion elemental analysis using a CHNS/O analyser (FlashSmart, Thermo Scientific). For each sample, 1.0-2.0 mg of dried powder was accurately weighed and analysed. The detection and quantification limits were 0.2 and 0.4 wt%, respectively, with an absolute uncertainty of ±0.4 wt%.

Water content of the samples was investigated by thermogravimetric analysis using a TGA instrument with a TA Instruments Discovery (Newcastle, UK). Approximately 10 mg of dry powder were placed in a platinum crucible and heated under a constant flow of inert gas (N₂, 50 mL min⁻¹). The temperature program consisted of an isothermal step at 30 °C for 20 min, followed by a linear heating ramp of 5 °C min⁻¹ up to 700 °C.

UV-vis spectra of the prepared suspensions were measured using a Shimadzu UV-2550 spectrophotometer and a YOKE Instrument spectrophotometer UV-1900 for the cation exchange measurements (CEC). 1.5 mL of 0.5 mg mL⁻¹ of the suspension was placed into quartz cuvettes, and all spectra were recorded from 190 to 900 nm with a 0.1 nm interval at 25 °C. The baseline was measured using ultrapure water or a NaCl (0.15 M) solution. Colloidal stability was assessed by measuring absorbance at 15 min intervals over a 15 h span, focusing on the maximum absorbance wavelength. The average oxidation state (AOS) of manganese in birnessite samples was determined through potentiometric titration method [6].

Emission spectra were collected on a Fluorolog-FL3-221 (Horiba Jobin-Yvon) spectrofluorimeter, equipped with three-slit double-grating excitation and emission monochromators with dispersions of 2.1 nm mm⁻¹ (1200 grooves mm⁻¹) and a red-sensitive Hamamatsu R928 photomultiplier tube as detector. Emission was monitored perpendicular to the excitation beam. Dilute samples (A₃₁₅ < 0.08) contained in a quartz Suprasil cell (Hellma) of 1-cm optical pathlength were excited by unpolarized light (λem = 315 nm) from a 450 W xenon CW (continuous-wave) lamp. The temperature was kept constant to 25.0 (2) °C by using a FL-1057 thermostated cell holder connected to a RE106 (Lauda) water circulator. Emission spectra acquired between 340 and 550 nm (Δλem = 1 nm, 0.1 s integration time) were corrected for source intensity (lamp and grating) and emission spectral response (detector and grating) by a standard correction curve provided by the manufacturer. For comparison purposes, their intensities were normalized with respect to the maximum of the Raman peak of water at 352 nm.

## 2.5. Determination of Cation Exchange Capacity (CEC)

The cation exchange capacity (CEC) of the Ns and nanofibers (Nf) was determined using the cobalt hexamine chloride method, as described in the French standard procedure AFNOR NF X31-130 (1999) [5]. Briefly, 150 mg of each sample was suspended in 20 mL of a 16.67 mM aqueous solution of [Co(NH₃)₆]Cl₃. The suspension was stirred for 1 h at 25 °C, then microfiltered through a 0.22 µm syringe filter to remove solid residues. The cobalt concentration before and after contact with the sample was measured by UV-vis spectrophotometry at 472 nm (ε₄₇₂ = 56.4 M⁻¹ cm⁻¹). The CEC was calculated from the
difference between initial and final cobalt concentrations, assuming complete exchange of interlayer cations with [Co(NH₃)₆]³⁺. To ensure accuracy, CEC values were normalized to the dry mass of the sample (after drying) to exclude water content and expressed in meq g⁻¹.

## 2.6. Reaction with GSH

The redox reactivity of birnessite-type MnO₂ nanomaterials toward reduced glutathione (GSH) was evaluated using MnO₂ suspensions (0.5 mg mL⁻¹) prepared in ultrapure water and homogenized via ultrasonication for 15 min. For each reaction, 10 mL of MnO₂ suspension was mixed with 5 mL of a freshly prepared GSH solution to achieve final GSH concentrations of 0.5, 2.0, and 4.0 mM. The pH of the reaction mixtures was adjusted to ~6.5 using 0.01 M HCl to simulate the mildly acidic tumor microenvironment.

Reaction kinetics were monitored over 60 min using a UV-vis spectrophotometer (200–800 nm, quartz cuvettes, 1-cm path length). Morphology-dependent reactions were tracked by measuring absorbance at characteristic λmax values: 367.5 nm for MnO₂ Ns and 357.5 nm for MnO₂ Nf, corresponding to Mn(IV)–O electronic transitions. The decrease in absorbance at these wavelengths served as a direct indicator of MnO₂ reduction by GSH. Spectra were recorded at 5-min intervals (0–60 min) for kinetic analysis.

The time-dependent decay of MnO₂ concentration was fitted to a pseudo-first-order exponential decay model (Eq. 3):

$$A(t) = A_0 e^{-kt} + A_{\infty} \quad (3)$$

where A(t) is the absorbance at time t, A₀ is the initial absorbance, k is the apparent first-order rate constant (min⁻¹), and A_∞ is the residual absorbance at infinite time. Non-linear regression was performed using the Levenberg–Marquardt algorithm, with constraints ensuring physically meaningful parameters (k > 0, 0 ≤ A_∞ ≤ A₀).

To confirm the reproducibility of the reactions, the experiment at the lowest GSH concentration was carried out in triplicate. The results demonstrate that the reaction is consistent, with the apparent rate constant varying by less than 10 %.

## 2.7. ICP-OES quantification of Mn²⁺ released upon reaction with GSH

Dissolved manganese produced by the redox reaction between MnO₂ and glutathione (GSH) was quantified using inductively coupled plasma–optical emission spectrometry (ICP-OES). MnO₂ samples were dispersed in deionized water at a concentration of 0.5 mg mL⁻¹ (total volume: 5.0 mL). GSH was added from a freshly prepared 100 mM aqueous stock solution to obtain final concentrations of 0, 0.5, 2.0, and 4.0 mM. The suspensions were gently shaken for 16 h at room temperature to allow the redox reaction to reach a quasi-steady state. After incubation, the suspensions were centrifuged, and the supernatants were filtered through 0.22 µm PTFE syringe filters to remove any remaining solid particles. The clear solutions were then acidified by adding 100 µL of concentrated HNO₃ (60 %) and analyzed by ICP-OES (Agilent 5110). Quantification was performed using an external calibration curve constructed from standard Mn²⁺ solutions. The resulting manganese concentrations are reported as [Mn²⁺] (mg mL⁻¹) as a function of the initial GSH concentration. In addition, the total manganese content of each sample was determined using the highest GSH concentration, for which complete reduction of MnO₂ was achieved.

## 2.8. Methylene Blue (MB) assay for ROS detection

The generation of hydroxyl radicals (OH*) by MnO₂ nanostructures was assessed using the MB degradation assay as a probe reaction. Reaction mixtures consisted of MB (10 µg mL⁻¹), H₂O₂ (10 mM), and MnO₂ nanoparticles (0.5 mM) suspended in ultrapure water. Reduced glutathione (GSH) was added at varying concentrations (0.5, 2, 5, and

10 mM) to induce redox cycling. All reactions were conducted in 25 mM sodium bicarbonate buffer (pH 7.4) at room temperature (~25 °C).

MB degradation was monitored over time by UV–vis spectroscopy, tracking the decrease in absorbance at 664 nm. Control experiments were performed in parallel under the following conditions:

* MB + H₂O₂ (no MnO₂)

* MB + MnO₂ (no H₂O₂)

The decay of MB absorbance at 664 nm was analyzed using a pseudo-first-order kinetic model (Eq. 4):

$$A' = A'_{0} e^{-k_{app} t} \quad (4)$$

where A' and A'₀ represent the absorbance at time t and at t = 0, respectively, and kₐpp is the apparent rate constant. The pseudo-first-order model was justified by the experimental conditions, where MB was used as a probe at low concentration, while H₂O₂, GSH, and MnO₂ were present in excess.

Reproducibility was ascertained by performing the experiment at a GSH concentration of 2 mM in triplicate. The results indicate consistent behavior, with the apparent rate constant varying by less than 10 %.

## 2.9. Fluorescence detection of hydroxyl radicals using terephthalic acid

Terephthalic acid (TA) was used as a selective fluorescence probe for hydroxyl radicals (OH*), since non-fluorescent TA is converted into the fluorescent 2-hydroxyterephthalic acid (HTA) upon reaction with OH* [19]. A TA stock solution (20 mM) was prepared by dissolving TA in 2 mM NaOH under ultrasonication until complete dissolution, then diluted to a final concentration of 0.5 mM in the reaction medium. For fluorescence measurements, 3.0 mL reaction mixture were prepared directly in quartz cuvettes (1-cm path length) containing MnO₂ samples (0.5 mg mL⁻¹), TA (0.5 mM), and the appropriate combination of H₂O₂ (10 mM final), and GSH (2 mM final). After mixing, the cuvettes were incubated for 6 h at room temperature in the dark to allow HTA formation. Fluorescence spectra were then recorded on a spectrofluorometer (excitation at 315 nm, emission collected between 350 and 550 nm). For each spectrum, the fluorescence intensity at the HTA maximum (λ ≈ 430–440 nm) was normalized to the intensity of the water Raman band (I<sub>Raman</sub>) recorded in the same spectrum, and the resulting ratio I<sub>max</sub> / I<sub>Raman</sub> was used for quantitative comparison between samples.

# 3. Results and discussion

## 3.1. Tetramethylammonium-birnessite characterization

Fig. 2a presents a TEM image of TMA-birnessite, revealing a mixture of crumpled exfoliated layers and stacked, clustered layers. The number of layers per tactoid depends on several factors, including the nature of the interlayer cation, which is determined by the base used during synthesis. For instance, when NaOH is employed, sodium cations occupy the interlamellar space, resulting in well-crystallized birnessite with a high degree of layer stacking [20]. The average number of layers per tactoid is governed by the strength of the interaction between the layers and the interlamellar cations: stronger interactions (typically observed with smaller hydrated cation radii) promote the formation of thicker stacks. In this study, the use of TMAOH introduced TMA⁺ into the interlamellar space. Due to the larger size of TMA⁺ vs Na⁺, the interlayer interactions are weaker, reducing the number of stacked layers per tactoid and sometimes enabling complete exfoliation [21]. This observation is further supported by dynamic light scattering (DLS) analysis (Fig. 2b), which shows a particle size distribution with three distinct peaks at 0.5 µm, 1.5 µm, and 2.6 µm. Notably, a significant fraction of particles (~0.5 µm) was detected, consistent with the presence of
<figure><img src="image_6.png" /><figcaption>Fig. 2. (a) TEM images, (b) hydrodynamic diameter at pH 9, (c) N₂ adsorption-desorption isotherm and (d) pore size distribution of TMA-birnessite.</figcaption></figure>

exfoliated or thinly stacked layers. The polydispersity index (PDI) of 0.361 indicates a relatively broad size distribution, in agreement with the heterogeneous morphology observed by TEM.

Fig. 2c displays the nitrogen adsorption-desorption isotherm of TMA-birnessite, which exhibits a type IVa profile with an H3 hysteresis loop according to the IUPAC classification, indicating the presence of a hierarchical porous structure combining mesopores (2–50 nm) and macropores (> 50 nm) [22]. The broad hysteresis loop observed between relative pressures of 0.45 and 0.99 (p/p⁰) indicates capillary condensation within a continuous porous network, encompassing both mesopores and macropores. This interpretation is further supported by the pore size distribution derived from the BJH method (Fig. 2d). The broad and heterogeneous pore size distribution can be attributed to the disordered and non-rigid stacking of birnessite layers. Specifically, smaller pore

diameters likely arise from constriction zones or contact points between adjacent layers, whereas larger pore diameters correspond to more open interlayer spaces. The specific surface area of the synthesized TMA-birnessite, as determined from the N₂ adsorption isotherm using the BET method, was found to be 129 ± 4 m² g⁻¹. It is worth noting that the specific surface area of birnessite can vary significantly depending on the synthesis method, typically ranging from approximately 50 to over 200 m² g⁻¹ [23,24]. In this study, the measured surface area of the TMA-birnessite sample suggests a relatively low degree of layer stacking within tactoids, which in turn indicates a comparatively low crystallinity.

X-ray diffraction (XRD) analysis was performed to determine the crystalline structure of the untreated TMA-birnessite powder with the resulting diffractogram revealing three distinct diffraction peaks at basal

<figure><img src="image_7.png" /><figcaption>Fig. 3. (a) XRD pattern and (b) Normalized Raman scattering spectrum of TMA-birnessite. * Artifacts resulting from the anti-diffusion blade during sample preparation.</figcaption></figure>
spacings of 0.954 nm, 0.478 nm, and 0.322 nm corresponding to the (001), (002), and (003) planes of birnessite respectively [18] (Fig. 3a). Comparison with the reference pattern of triclinic Na-birnessite (Table S1; ICDD 00-043-1456) reveals similar peaks, with a systematic shift of the basal (001) peaks to lower 2θ due to TMA⁺ intercalation. Specifically, the *d*₀₀₁ peak of Na-birnessite appears at 0.713 nm [25], while it is located at 0.954 nm for TMA-birnessite.

The observed shift in *d*₀₀₁ reflects the larger interlayer spacing caused by the bulkier size and greater hydration shell of TMA⁺ relative to Na⁺. The intense (001) reflection observed in TMA-birnessite suggests that some Ns remain aggregated into stacked, tactoid-like structures, likely due to incomplete delamination during lyophilization. Furthermore, the asymmetrical reflections assigned to the (100) and (110) planes indicate a partial loss of crystallinity [15], which may arise from structural disorder induced by the intercalation of bulky TMA⁺ ions.

To confirm the triclinic symmetry of TMA-birnessite, Raman spectroscopy was employed. The spectrum presented in Fig. 3b exhibits characteristic Raman features of manganese oxides in the 700–400 cm⁻¹ range, with all observed bands listed in Table S2. Key bands include a peak at 637 cm⁻¹, attributed to the symmetric Mn–O stretching vibration of MnO₆ octahedra, and an intense band at 567 cm⁻¹, corresponding to the Mn–O stretching vibration in the basal plane of MnO₆ sheets. Another significant band at 488 cm⁻¹ is associated with the Mn–O–Mn bending vibration [3]. The narrow band at 567 cm⁻¹ is indicative of triclinic birnessite [6]. However, the Raman spectrum of this sample exhibits slight differences compared to that reported by Boumaiza et al. [6], particularly in the relative intensity of this band.

This discrepancy suggests a lower crystallinity for the birnessite synthesized in the presence of TMAOH, potentially due to a reduced number of stacked layers along the c-axis and limited lateral extension in the (a, b) plane.

The average oxidation state (AOS), determined by potentiometric titration [25,26], is 3.62. This value reflects a mixed-valence state arising from the coexistence of Mn(IV) and Mn(III), which is characteristic of triclinic birnessite-type structures. The measured AOS falls within the typical range of 3.5–3.7 reported for such materials [6].

The surface of TMA-birnessite was investigated by X-ray photoelectron spectroscopy (XPS). The overview XPS spectrum (Fig. 4a) shows core-level photoelectron of key elements of the TMA-birnessite namely manganese, oxygen, carbon and nitrogen while the presence of silicon is attributed to the support material used during the XPS analysis.

The O1s spectrum of MnO₂ (Fig. 4b) exhibits a main peak at 530.2 eV and a shoulder at higher binding energies (532.1 eV). These values are consistent with previous reports, which assign binding energies of 530.8–530.9 eV to oxygen atoms bonded to Mn [26]. The O1s shoulder at 531–532 eV likely arises from both structural hydroxyl groups (Mn–OH) and physisorbed water, whose binding energies are very close, making it difficult to clearly separate these contributions by XPS [27]. In our case, thermogravimetric analysis indicates a low water content for the nanosheets (≈3.2 wt%, Table S3), suggesting that the observed shoulder likely arises from a combination of both surface hydroxyl groups and the contribution of physisorbed water.

To distinguish the oxidation states of manganese, the Mn3s peak is analyzed (Fig. 4c). This peak consists of two components arising from

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** XPS spectra of TMA-birnessite: (a) survey spectrum, and fitted curves of (b) O1s, (c) Mn3s and (d) Mn3p peaks.</figcaption></figure>
the coupling between the 3s electron and the 3d electrons. According to Biesinger *et al.* [28], the energy separation between these two components enable to assign the Mn oxidation state. A difference of 5.9, 5.5, or 4.8 eV corresponds respectively to Mn(II), Mn(III), or Mn(IV). The TMA-birnessite shows a value of 4.88 eV, indicating that the oxidation state of manganese is close to 4. Ilton *et al.* [29] suggested that the Mn3p signal can be reliably used to determine the distribution of Mn(II), Mn(III), and Mn(IV) on the surface. Fitting the Mn3p peak (Fig. 4d) reveals that the surface consists of 59 % Mn(IV) and 41 % Mn(III), yielding an average oxidation state (AOS) of 3.59. This calculated AOS is consistent with the value of 3.62 obtained from potentiometric titration, confirming the presence of a mixed-valence state characteristic of triclinic birnessite.

The total nitrogen content (4.2 wt% N, determined by elemental CHN analysis), the water content (3.2 wt%, determined from TGA), and the total manganese concentration (40.0 wt% determined by ICP-OES) were combined to refine the chemical formula of the pristine triclinic TMA-birnessite (Table S2). Obtained values correspond to a TMA/MnO₂ molar ratio of x ≈ 0.41 and a hydration degree of 0.24 H₂O per MnO₂ unit, leading to an overall composition (TMA)₀.₄₁MnO₂·0.24H₂O (Table S3). This composition implies a negative layer charge balanced by TMA⁺ cations, originating from partial Mn(IV) substitution by Mn (III), which can be expressed as (TMA)₀.₄₁MnIV₀.₅₉MnIII₀.₄₁O₂·0.24H₂O. This formulation is in excellent agreement with XPS data (Mn(IV)/Mn (III) = 59/41) and gives an average oxidation state (AOS) of 3.59, close to the potentiometrically determined 3.62. The theoretical cation-exchange capacity (3.5 meq g⁻¹) is in reasonable agreement with the experimental CEC (3.86 meq g⁻¹), with the remaining ~10 % difference attributed to exchangeable surface hydroxyl groups (–OH) at the layer edges.

To assess the colloidal stability of untreated birnessite in ultrapure water at pH 7.4, the change in absorbance of the suspension over time was monitored [30]. At 7.4 pH, the zeta potential of TMA-birnessite is approximately –10 mV. Generally, a zeta potential greater than ±30 mV is considered necessary to ensure strong colloidal stability through sufficient electrostatic repulsion between particles [31]. However, it has been reported that colloidal stability can also be maintained at lower zeta potential values when other stabilizing mechanisms such as steric hindrance, solvation effects, or specific surface conditions are at stake [32]. In this study, despite the relatively low zeta potential, the TMA-birnessite particles exhibited good colloidal stability, suggesting that factors beyond purely electrostatic repulsion contribute significantly to maintaining dispersion stability under the tested conditions. When TMA-birnessite is dispersed in a 0.15 M NaCl aqueous solution, a decrease in relative absorbance from 100 % to 20 % in 14 h is observed

(Figure 5a2). The NaCl solution mimics the physiological environment where similar ionic effects are likely to influence the stability of MnO₂ dispersions [33]. It is well known that colloidal stability decreases as the ionic strength of the suspension increases [34]. Indeed, the cations in the solution screen the negative charge of the particles, thereby preventing their electrostatic repulsion. The particles can then come close enough for Van der Waals interactions to occur, thus leading to the agglomeration of the particles and their subsequent sedimentation. Additionally, the interaction between Na⁺ and the birnessite layers is stronger than that between TMA⁺ and the layers which increases aggregation. These observations indicate that while birnessite can remain stable in ultrapure water, the ionic strength found in biological fluids may compromise its colloidal stability.

When the medium is changed to phosphate-buffered saline (PBS) at the same pH, the TMA-birnessite undergoes rapid sedimentation that occurs almost instantaneously (Fig. 5b). The more complex ionic composition of PBS which contains K⁺ in addition to Na⁺ exacerbates the reduction in electrostatic repulsions and leads to swift aggregation and sedimentation of the birnessite particles. Owing to its larger ionic radius, K⁺ has a lower charge density than Na⁺. Consequently, the hydration structure around K⁺ is different from that around Na⁺, with K⁺ having weaker ion-water interactions due to lower charge density, which facilitates stronger interactions with the birnessite layers compared to the more strongly hydrated Na⁺. These findings underscore the importance of modifying the surface of birnessite to enhance its colloidal stability in physiological media, which is essential for successful biomedical applications.

## 3.2. Effect of ultrasonication duration on TMA-birnessite

The morphology of birnessite particles as a function of the ultrasonication duration was studied using TEM. Fig. 6 presents the particles before and after 10, 20, 25, and 30 min of ultrasonication. Progressive changes in the structure of the particles are observed as the ultrasonication time increases. Before ultrasonication (Fig. 6a), the birnessite particles exhibit both agglomerated and delaminated layers. After 10 min of ultrasonication (Fig. 6b), increased dispersion of the agglomerates is observed with the presence of uncrumpled Ns. The energy provided by the ultrasounds appears to overcome the interparticle interactions leading to the separation of the Ns. After 20 min of ultrasonication (Fig. 6c), an initial curling of the Ns is observed indicated by more defined structures and curvatures. This suggests that the energy supplied by the ultrasounds is sufficient to induce mechanical stress leading to deformation and initial curling of the Ns. The extent of the Ns observed in Fig. 6c is less than that in Fig. 6b indicating the onset of

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Evolution of the relative absorbance over time for TMA-birnessite dispersed in ultrapure water at pH 7.4 (a1) and in 0.15 M NaCl solution (a2). (b) Photographs of TMA-birnessite mixtures in ultrapure water and in PBS after 5 min of mixing.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** TEM images of TMA-birnessite (a) before and after (b) 10 min, (c) 20 min, (d) 25 min, and (e) 30 min of ultrasonication. (f) High-resolution TEM of nanofibers formed after 30 min, showing lattice spacing of 0.34 nm (003) plane. Scale bars: 500 nm (a–e), 5 nm (f).</figcaption></figure>

sheet fragmentation due to the ultrasound energy. After 25 min of ultrasonication (Fig. 6d), the particles exhibit a more aligned structure suggesting the continuation of either curling or anisotropic fragmentation of the sheets. Such observations have been previously noticed in our group in the case of titanate nanostructures [35,36].

Taken together, these TEM observations suggest a stepwise, cavitation-driven transformation mechanism from stacked nanosheets to one-dimensional nanofibers. At short sonication times ($\le$ 10 min), acoustic cavitation mainly disperses the large aggregates and partially delaminates the stacked TMA-birnessite layers, producing more isolated and flatter nanosheets. Between 20 and 25 min, repeated bubble collapse generates local stresses that concentrate at defects and sheet edges, initiating bending and curling of the thinnest flakes. Under continued ultrasonication, this anisotropic mechanical stress preferentially fragments the sheets along the weakest crystallographic directions, leading to elongated ribbons. At 30 min, these ribbons further align and assemble by oriented attachment into dense bundles of nanofibers with lengths of ~500 nm and diameters of 10–15 nm (Fig. 6e–f). This scenario explains the coexistence of nanosheets and nanofibers at 25 min and the near-complete conversion to nanofibers after 30 min of sonication.

After 30 min of ultrasonication, the TEM image (Fig. 6e) shows only elongated structures. This could be the result of continuous curling or anisotropic fragmentation leading to a morphology radically different from the initial state. Fig. 6f shows that these are not curled sheets. A curled structure would resemble nanotubes with walls and a central channel [35,36]. Instead, the image shows 500-nm long rods (Fig. 6e), the width of which is approximately 15 nm, and a lattice spacing of 0.34 nm corresponding to the (003) plane of birnessite. However, this image shows that the rods are darker than the individual sheets in Figs. 6b and 6c. This suggests that at a certain degree of division, the oxide sheets fold forming bent rods that resemble Nf. The term "nano-whisker" is often used in literature to describe this morphology due to its resemblance to elongated whisker-like structures [37].

The anisotropic fragmentation of the sheets is consistent with the triclinic birnessite structure, which consists of alternating chains of Mn (III)O₆ and Mn(IV)O₆ octahedra. Because Mn(III) octahedra are Jahn-Teller distorted, the corresponding Mn(III)O₆ chains are mechanically weaker and more prone to cleavage than the Mn(IV)O₆ chains. Cavitation-induced stresses therefore promote crack propagation along these Mn(III)-rich directions, favoring the formation of elongated ribbon-like fragments that subsequently fold and assemble into nanofibers.

To assess the impact of ultrasonication on the structure and

crystallinity of TMA-birnessite, X-ray diffraction and Raman spectroscopy were performed. The XRD patterns (Fig. 7 A) show that all samples retain the birnessite phase throughout 0–30 min of ultrasonication. Small additional features appear around $2\theta \approx 40^{\circ}$ and $\approx 75^{\circ}$, but these could not be consistently assigned to any known Mn-oxide phase or impurity. The (001) reflections of birnessite exhibit progressive broadening and a noticeable decrease in intensity as the sonication time increases. This peak broadening reflects a continuous reduction in coherent crystallite size, along with the generation of internal lattice strain. These structural parameters were quantified using Williamson-Hall (W-H) analysis [38] and results are presented in Table 1. The individual W-H plots for each sample are provided in Supplementary Information (Figure S1). At 0 min, the average crystallite size (D) was 9.6 nm, with a low microstrain rate ($\epsilon$) of 0.2%, indicating a well-ordered layered structure with minimal internal defects. After 20 min of ultrasonication, the crystallite size decreased markedly to 6.6 nm, accompanied by a sharp increase of the microstrain rate to 1.4%. This concomitant decrease in crystallite size and increase in microstrains indicate progressive fragmentation of the initial nanosheets. Fragmentation is driven by cavitation, which produces localized shear stresses and high-energy collisions that generate lattice defects. Prolonging ultrasonication to 30 min further reduced the crystallite size to 3.1 nm. Interestingly, the microstrain rate remained nearly unchanged, suggesting a partial relaxation of internal stresses at this stage. These structural changes are consistent with the morphological evolution observed by TEM (Fig. 6), where MnO₂ progressively transforms from well-defined Ns to fragmented domains and, ultimately, assembles into one-dimensional Nf under prolonged ultrasonication. The sharp increase of the microstrain rate ($\epsilon$) indicates that cavitation not only reduces the crystallite size but also generates a substantial structural distortions.

Raman spectra recorded for TMA-birnessite before and after 20 and 30 min of ultrasonication shows nearly identical profiles, indicating that sonication does not change the triclinic birnessite framework.

Next, the effect of ultrasonication on the colloidal size and porous texture of TMA-birnessite was investigated. Hydrodynamic size distributions are reported in Table S4 and Fig. 8a–b, while textural properties are shown in Fig. 8c–d. The untreated sample exhibited a multimodal distribution, with one population corresponding to primary particles and other peaks associated with larger agglomerates; the broadness of the peaks reflects a wide size distribution. With increasing ultrasonication time, the second peak progressively disappeared, and the distribution shifted towards smaller sizes (Fig. 8b), highlighting a clear deagglomeration effect. After 30 min of treatment, the distribution
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** (A) XRD pattern and (B) normalized Raman scattering spectra of TMA-birnessite: (a) before sonication, (b) after 20 min, and (c) after 30 min of sonication. * Artifacts resulting from the anti-diffusion blade during sample preparation.</figcaption></figure>

<caption>**Table 1**</caption>

Crystallite size (D) and microstrain level (ε) of MnO₂ as a function of ultrasonication time, determined by Williamson-Hall analysis. Errors represent standard deviations from linear fitting.

<table><thead><tr><td>Ultrasonication time (min)</td><td>Crystallite size D (nm)</td><td>Microstrains ε (%)</td></tr></thead><tbody><tr><td>0</td><td>9.6 ± 2.1</td><td>0.2 ± 0.1</td></tr><tr><td>20</td><td>6.6 ± 3.2</td><td>1.4 ± 0.2</td></tr><tr><td>30</td><td>3.1 ± 0.9</td><td>1.3 ± 0.2</td></tr></tbody></table>

became monomodal and significantly narrower, demonstrating that ultrasonication effectively breaks down agglomerates and produces a more homogeneous population of smaller particles. This trend is confirmed quantitatively (Table S1): the average hydrodynamic

diameter decreased from 544 to 173 nm, while the polydispersity index (PdI) dropped from 0.36 to 0.24, reflecting a narrower distribution. Such reduction in size and improvement in uniformity are advantageous for applications requiring precise control of particle dimensions.

The nitrogen adsorption-desorption isotherm of the ultrasonicated sample (Fig. 8c) corresponds to type IVa with H1 or H2b hysteresis according to the IUPAC classification [22], indicating a well-defined mesoporous structure. Following ultrasonication, the specific surface area increased to 142 m² g⁻¹, representing a 10% enhancement compared to the untreated TMA-birnessite. The narrower hysteresis loop, observed between p/p⁰ = 0.55 and 0.95, reflects capillary condensation within a mesoporous network centered around 10 nm, as confirmed by the BJH pore size distribution (Fig. 8d). These results—increased surface area and refined mesoporous

<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Evolution of (a) particles size distribution, error bars represent ± standard deviation (n = 3), and (b) average hydrodynamic size as a function of ultrasonication duration. (c) N₂ adsorption-desorption isotherm of TMA-birnessite after 30 min of ultrasonication, and (d) BJH adsorption dV/dlog(D) pore volume after 30 min of ultrasonication.</figcaption></figure>
structure—demonstrate a significant improvement in the textural properties of the material after ultrasonic treatment.

To better understand the effect of ultrasonication on the surface properties of birnessite, XPS analysis was performed on the sample subjected to 30 min of ultrasonication. Fig. 9a shows the XPS spectrum of TMA-birnessite after 30 min of ultrasonication, along with the fitted curves for the O1s (Fig. 9b), Mn3s (Fig. 9c) and Mn3p peaks (Fig. 9d). The survey spectrum (Fig. 9a) aligns with that of the untreated sample, confirming the presence of the same elements constituting TMA-birnessite. Table 2 presents the elemental composition of both untreated and treated birnessite samples. In untreated birnessite, the O/Mn ratio determined by XPS is 1.8, close to the theoretical value of 2 for MnO₂, whereas sonication causes an increase of this ratio. This observation is consistent with the fragmentation of the birnessite layers. During the fragmentation of a layer, each broken Mn–O–Mn bond forms two Mn–OH groups at the newly formed edges in the presence of water:

$$Mn-O-Mn + H_2O = 2 Mn-OH$$

This can be observed in the O1s spectrum (Fig. 9b) where the percentage of oxygen in MnO₂ decreased while the proportion of OH groups, corresponding to structural –Mn–OH groups and adsorbed water, increased from 30 % before sonication to 41 % after 30 min of sonication. Thermogravimetric analysis nevertheless shows a slightly varying water content for the nanosheets and nanofibers (3.2 and 2.8 wt %, respectively; Table S3), suggesting that the increase of the 531–532 eV envelope cannot be ascribed solely to a higher amount of physically adsorbed water. Furthermore, after sonication, Mn(II) appears in the sample (Fig. 9d) leading to a decrease in the AOS compared to the untreated sample. The AOS calculated through the fitting of the Mn3p peak for the sample subjected to 30 min of ultrasonication is 3.53, which is 1.7 % lower than the AOS of 3.59 found by the same method for

<table><caption>**Table 2**
Molar percentages of treated and untreated birnessite samples.</caption><thead><tr><th>TMA-birnessite</th><th>C</th><th>O</th><th>N</th><th>Mn</th><th>O/Mn</th><th>N/Mn</th></tr></thead><tbody><tr><td>before ultrasonication</td><td>25.0</td><td>46.5</td><td>3.0</td><td>25.5</td><td>1.8</td><td>0.1</td></tr><tr><td>after 30 min of ultrasonication</td><td>23.3</td><td>52.4</td><td>2.1</td><td>22.2</td><td>2.4</td><td>0.1</td></tr></tbody></table>

the untreated sample. The AOS determined by potentiometric titration also decreased from 3.62 to 3.53. The appearance of Mn(II), the decrease of Mn(III) and the increase of Mn(IV) (Fig. 9d) suggest that sonication leads to a disproportionation reaction, creating vacancies in the structure and releasing Mn²⁺ ions (Mn²⁺ is most likely not accommodated within the layer [6]). Furthermore, the energy difference between the two Mn3s peaks (Fig. 9c) increased from 4.88 to 4.94 eV, confirming that the AOS tends towards values closer to 3. Therefore, we can conclude that the AOS decreases as a function of ultrasonication duration.

XPS analysis detected the presence of nitrogen in the nanofibers (Table 2); however, elemental analysis indicated that the content was below the detection limit of 0.2 wt% (Table S2). Considering all results, it was not possible to determine a definitive chemical formula for the nanofibers. It can only be inferred that the fibers contain Mn(IV), Mn (III), and vacancies, and that the released Mn²⁺ likely contributes to balancing their negative charge. [6] The experimental CEC increase from 3.86 to 4.15 meq g⁻¹ upon sonication should result from the higher density of OH⁻ surface groups for Nf as compared to Ns. Indeed, the CEC comes from the negative charges within the layers but also from the OH groups on the edge of the layers.

To gain further insights into the effect of ultrasonication on the surface of birnessite, zeta potential was measured (Fig. 10a). After 30 min of sonication, the value of zeta potential, at pH 7 becomes more negative (from –10 to –35 mV), while the isoelectric point shifts from

<figure><img src="image_13.png" /><figcaption>**Fig. 9.** XPS spectra of TMA-birnessite after 30 min of ultrasonication: (a) survey spectrum and fitted curves of (b) O1s, (c) Mn3s, and (d) Mn3p peaks.</figcaption></figure>
5.5 to 2.2. The increase in the absolute value of the zeta potential indicates that sonication produces nanoparticles that repel each other more effectively, suggesting improved colloidal stability [39]. There are numerous variations in the isoelectric point values measured for manganese oxides. This parameter depends on several factors such as the synthesis method, the crystalline structure, the oxidation state of manganese, the coordination with oxygen, and the formation of manganese oxide complexes at the interface between the oxide and the aqueous solution [40]. In the case of TMA-birnessite after sonication, a modification in the oxidation state of manganese, an increase in the density of hydroxyl groups and the presence of Mn²⁺ likely complexed at the surface have been observed. It is also probable that an increase in the concentration of Mn(III)-OH at the layer edges occurred due to anisotropic fragmentation, which promotes layer breakage along of the Mn (III)O₆ chains. All these phenomena of course influence both the zeta potential and the isoelectric point of birnessite.

To evaluate the effect of ultrasonication duration on the colloidal stability of TMA-birnessite, a kinetic study was conducted by measuring absorbance over time for different samples: before ultrasonication and after treatment for 10, 20, and 30 min. This analysis was performed in two media: ultrapure water at pH 7.4 and 0.15 M aqueous NaCl solution. Fig. 10b illustrates the absorbance variation of birnessite nanoparticle suspensions in ultrapure water at pH 7.4 over 15 h. The graph compares the absorbance variations over time for samples subjected to different ultrasonication durations. All suspensions display good stability although TMA-birnessite samples after ultrasonication exhibit higher colloidal stability. It is relevant to recall the 3-fold higher zeta potential measurement for the sample subjected to a 30-min sonication compared to the untreated sample. This increase of the absolute zeta potential is associated with enhanced colloidal stability due to greater repulsion between particles. However, a 50 % decrease in absorbance is only observed after 2 h in the case of NaCl (Fig. 10c). Indeed, reducing the ionic strength of the medium mitigates the salting-out effect and favors the dispersion of the nanoparticles, which is essential for successful biomedical applications requiring long-term colloidal stability. In both more complex systems (×PBS 1 × and BSA-containing solutions), nanosheets and nanofibers rapidly formed large aggregates, resulting in sedimentation within minutes. This observation underscores the critical role of surface modification in achieving steric stabilization.

### 3.3. Kinetic analysis and morphological impact on the reduction of birnessite by glutathione in acidic medium

The redox interaction between birnessite (MnO₂) and reduced glutathione (GSH) was investigated under mildly acidic conditions (pH 6.5), selected to reproduce the tumor microenvironment, which is typically characterized by acidity and abnormally elevated intracellular GSH levels in the range of 2–10 mM [41]. It is well established that MnO₂ is efficiently reduced by GSH, producing Mn²⁺ and oxidized

glutathione (GSSG), according to the following reaction [8]:

$$MnO_2 + 2GSH + 2H_3O^+ = Mn^{2+} + GSSG + 4H_2O$$

The UV–vis absorption spectra of birnessite-type MnO₂ Ns (before treatment) and Nf (after 30 min of ultrasonication treatment) exhibited distinct signatures characteristic of their morphological differences (Figure S2). Ns showed a maximum absorbance at 369 nm, while Nf exhibited its maximum at 358 nm. This 11 nm hypochromic shift indicates a change in the electronic environment of manganese centers related to morphological features, likely due to differences in surface facet exposure and crystallite organization [8]. The higher optical density found for Ns (ratio of 1.3) reflects their compact two-dimensional structure, providing a robust foundation for morphology-specific kinetics, which is essential for accurate quantitative analysis.

Kinetic measurements of MnO₂ reduction by GSH were conducted at physiologically relevant concentrations (0.5–4.0 mM) over a period of 60 min. The resulting kinetic profiles were well described by a pseudo-first-order model (Fig. 11), with R² values exceeding 0.94, and residuals randomly distributed around zero (Figure S3), further confirming the validity of the model. The selected model, expressed in Eq. 3 includes a residual absorbance term (A∞) representing the fraction of MnO₂ inaccessible to reduction, possibly due to surface heterogeneity or steric hindrance.

The apparent rate constants (k) clearly depend on GSH concentration (Table 3), increasing for both Ns and Nf as the concentration rises. A log-log plot of k versus GSH concentration revealed that k is proportional to [GSH]¹.⁰.⁷ (Figure S4), suggesting a complex mechanism involving surface adsorption and diffusion-limited processes, which are typical features of heterogeneous solid–liquid reactions [42] (Table 4).

As summarized in Table 3, the apparent rate constants (k) for GSH reduction increased with concentration for both morphologies, but Nf consistently outperformed Ns, highlighting their superior catalytic efficiency, with an enhancement factor of approximately 1.5, corresponding to the ratio of the rate constants. This behavior is attributed to their fibrillar structure, which provides a greater surface area and more accessible reactive sites compared to the layered Ns morphology. Residual absorbance values (A∞) decreased with increasing GSH concentration for both Ns and Nf. These results suggest that higher GSH levels enhance accessibility to previously unavailable Mn(IV) sites, possibly through partial surface restructuring or conformational changes in the material [43].

The extent of MnO₂ reduction by GSH was directly quantified by measuring the dissolved Mn concentration after 16 h of contact between MnO₂ and GSH for both morphologies (Figure S5). In the absence of GSH, the amount of Mn detected in solution is very low (0.02 mg mL⁻¹), confirming that TMA-birnessite is stable in water and does not undergo significant spontaneous dissolution. When GSH is added, the Mn²⁺ concentration increases monotonically with the GSH one, from about

<figure><img src="image_14.png" /><figcaption>Fig. 10. (a) Variation of the zeta potential of birnessite nanoparticles as a function of pH, error bars represent ± standard deviation (n = 3), (b) Evolution of the relative absorbance at 369 nm over time for untreated birnessite and those treated by sonication for 20 and 30 min, dispersed in ultrapure water at pH 7.4 and (c) in a 0.15 M NaCl solution.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 11.** Time-dependent decrease in absorbance at 369 nm for the reduction of MnO₂ by GSH at different concentrations (0.5, 2.0, and 4.0 mM) fitted to a pseudo-first-order kinetic model. (a) Nanosheets (Ns). (b) Nanofibers (Nf).</figcaption></figure>

**Table 3**

Kinetic parameters for the reduction of MnO₂ nanosheets and nanofibers by GSH at different concentrations, including rate constants ($k$), residual absorbances ($A_{\infty}$), and coefficients of determination ($R^2$).

<table><thead><tr><th>[GSH] (mM)</th><th>Morphology</th><th>k (min<sup>-1</sup>)</th><th>A<sub>∞</sub></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>0.5</td><td rowspan="3">Nanosheets</td><td>0.017</td><td>0.12</td><td>0.96</td></tr><tr><td>2.0</td><td>0.045</td><td>0.08</td><td>0.98</td></tr><tr><td>4.0</td><td>0.061</td><td>0.05</td><td>0.99</td></tr><tr><td>0.5</td><td rowspan="3">Nanofibers</td><td>0.028</td><td>0.10</td><td>0.94</td></tr><tr><td>2.0</td><td>0.069</td><td>0.06</td><td>0.97</td></tr><tr><td>4.0</td><td>0.097</td><td>0.01</td><td>0.98</td></tr></tbody></table>

**Table 4**

Apparent rate constants ($k_{app}$) and correlation coefficients ($R^2$) for the MB degradation reaction catalyzed by MnO₂ nanosheets and nanofibers at different GSH concentrations.

<table><thead><tr><th rowspan="2">[GSH] (mM)</th><th colspan="2">Nanosheets</th><th colspan="2">Nanofibers</th></tr><tr><th>k<sub>app</sub> (min<sup>-1</sup>)</th><th>R<sup>2</sup></th><th>k<sub>app</sub> (min<sup>-1</sup>)</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>2</td><td>0.067</td><td>0.990</td><td>0.063</td><td>0.992</td></tr><tr><td>4</td><td>0.031</td><td>0.999</td><td>0.028</td><td>0.998</td></tr><tr><td>8</td><td>0.014</td><td>0.978</td><td>0.014</td><td>0.978</td></tr><tr><td>10</td><td>0.010</td><td>0.947</td><td>0.010</td><td>0.965</td></tr></tbody></table>

0.09 mg mL⁻¹ at 0.5 mM GSH to 0.20 mg mL⁻¹ at 4 mM GSH for nanosheets, and from 0.10 to 0.21 mg mL⁻¹ for nanofibers. For each GSH concentration, Nf systematically released slightly more Mn²⁺ than Ns.

### 3.4. Detection of hydroxyl radicals (OH*) using MB assay

The ability of MnO₂ nanostructures to generate reactive oxygen species (ROS) in the presence of GSH was assessed by monitoring the MB degradation. MB exhibits two main absorption bands, with a dominant peak at 664 nm and a secondary shoulder at 610 nm (Figure S6). Hydroxyl radicals attack the π-delocalized system of MB, disrupting the conjugation, which is manifested by a decrease in absorbance at 664 nm and discoloration of the solution visible by the naked eye. The absorbances at 664 nm recorded under different conditions after 2 min of contact time showed that MB remained stable in the presence of H₂O₂ or MnO₂ alone (Fig. 12), confirming negligible spontaneous degradation. In contrast, when both H₂O₂ and GSH were combined with MnO₂, a marked decrease in MB absorbance at 664 nm was observed, demonstrating the catalytic generation of hydroxyl radicals. The comparative plot at λ = 664 nm highlights that Ns induced a stronger bleaching effect than Nf at all tested GSH concentrations. Moreover, increasing the GSH concentration from 0.5 to 10 mM progressively reduced the degradation

<figure><img src="image_16.png" /><figcaption>**Fig. 12.** Methylene blue (MB) absorbance at 664 nm measured after 2 min of reaction at various conditions. Condition: A = MB + H₂O₂; B = MB + MnO₂; C = MB + H₂O₂ + MnO₂. Bars to the right of C correspond to C + GSH at the indicated concentrations (0.5–10 mM). Lower absorbance indicates greater MB bleaching.</figcaption></figure>

of MB, consistent with a concentration-dependent inhibition effect.

To further analyze the kinetics, the decay of MB absorbance was monitored over time. It was observed that MB degraded faster at low GSH concentrations (2–4 mM), whereas higher ones (8–10 mM) markedly slowed down the process (Figure S7a). This behavior can be rationalized by the dual role of GSH. In this experiment, at low GSH concentrations, MnO₂ reacts with GSH to produce Mn²⁺, which converts H₂O₂ into hydroxyl radicals that attack MB. In contrast, at high GSH concentrations, excess GSH scavenges the hydroxyl radicals, preventing them from attacking MB. This dual role of GSH as both an activator and an inhibitor of ROS generation has also been reported for similar MnO₂-based systems [13]. The pseudo-first-order plots of ln(A(t)/A₀) versus time (Figure S7b) exhibited a good linearity (R² > 0.97), confirming the
validity of the kinetic model. The apparent rate constants ($k_{app}$) are summarized in Table 3. As expected, $k_{app}$ decreases with increasing GSH concentration, while both morphologies exhibit approximately the same $k_{app}$ at a given GSH concentration (the difference does not exceed 10 %). Despite their markedly different morphologies and surface properties, Ns and Nf show similar catalytic behavior, with a slight advantage observed for the nanosheets. The degradation of MB proceeds through a sequence of three reactions:

$$MnO_2 + 2GSH + 2H_3O^+ = Mn^{2+} + GSSG + 4H_2O$$

$$Mn^{2+} + H_2O_2 + H_3O^+ = Mn(IV) + OH^* + 2H_2O$$

$$MB + OH^* = \text{products}$$

It is possible that the rate-limiting step does not involve birnessite directly, so its morphology may not significantly affect the kinetics of MB degradation.

To further confirm that methylene blue bleaching arised from hydroxyl radical generation rather than from simple dye adsorption on MnO₂, we complemented the MB assay with the one using the terephthalic acid (TA) fluorescence probe (Fig. 13a). TA alone and TA + H₂O₂ produced only a very weak background signal, indicating that spontaneous HTA formation is negligible in the absence of MnO₂. Similarly, mixing TA with Ns or Nf in the absence of H₂O₂ led to only a modest increase of the normalised emission at ca. 435 nm, confirming that MnO₂ by itself does not generate significant amounts of OH* under these conditions.

In contrast, combining MnO₂ with H₂O₂ resulted in a clear enhancement of the HTA fluorescence, both in the presence of nanosheets and nanofibers, showing that MnO₂ catalyses H₂O₂ decomposition into reactive oxygen species. The signal increased much more strongly when GSH (2 mM) was added together with H₂O₂, consistent with a GSH-driven reduction of Mn(IV/III) to Mn²⁺ followed by a Fenton-like reaction with H₂O₂ that amplifies OH* production. Quantitatively, the normalised values (Fig. 13b) were systematically higher for nanosheets than for nanofibers in the presence of H₂O₂ and GSH, indicating that Ns behave as more efficient OH* generators than Nf under identical experimental conditions.

Overall, these TA fluorescence results are fully consistent with the MB degradation data and reinforce our conclusion that nanofibers are more efficient GSH scavengers, whereas nanosheets act as slightly more

potent hydroxyl-radical amplifiers in the MnO₂/H₂O₂/GSH redox system.

## 4. Conclusion

Ultrasonication of triclinic TMA-birnessite drives a controlled morphological transition from stacked Ns to one-dimensional Nf, yielding smaller particles with higher absolute zeta potentials and lower isoelectric points, and thus markedly improves colloidal stability in ultrapure water. In comparison to Ns, Nf reduce glutathione approximately 1.5 times faster, enabling more efficient intracellular antioxidant depletion, whereas particles of both morphologies catalyze hydrogen peroxide decomposition, producing reactive oxygen species (ROS), such as hydroxyl radicals, the nanosheets appear to perform better than the nanofibers. According to these morphology-dependent properties, ultrasonicated birnessite appears to be a promising candidate for dual-function cancer therapies, combining GSH depletion to sensitize tumor cells and controlled ROS generation for oxidative damages. Future work will focus on *in vitro* and *in vivo* validation and surface functionalization (e.g., polyethylene glycol coating) to further enhance physiological stability, minimize aggregation, and fine-tune the balance between antioxidant depletion and radical production. These advances will position MnO₂ nanostructures as versatile agents for drug delivery, theranostics, and precision cancer therapy.

### CRediT authorship contribution statement

**Mahran Bechetnia:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Conceptualization.
**Oumaima Hajlaoui:** Validation, Methodology, Investigation. **Julien Boudon:** Writing – review & editing, Validation, Supervision. **Michaële Herbst:** Validation, Methodology, Investigatio. **Rémi Chassagnon:** Investigation, Methodology, Validation. **Michel Meyer:** Investigation, Writing – review & editing. **Nadine Millot:** Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization. **Latifa Bergaoui:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Conceptualization.

<figure><img src="image_17.png" /><figcaption>Fig. 13. (a) Normalised fluorescence spectra of terephthalic acid (TA 20 mM) after 6 h incubation under different conditions: TA, TA + H₂O₂, TA + MnO₂ nanosheets (NS), TA + MnO₂ nanofibers (NF), TA + NS + H₂O₂, TA + NF + H₂O₂, TA + NS + H₂O₂ + GSH (2 mM) and TA + NF + H₂O₂ + GSH (2 mM). Spectra were recorded at $\lambda_{exc}$ = 315 nm and normalised to the intensity of the water Raman band. (b) Corresponding normalised maximum fluorescence intensity at the HTA emission maximum ($I_{max}$ / $I_{Raman}$) highlighting the influence of the reaction conditions and MnO₂ morphology (NS vs NF) on OH* generation.</figcaption></figure>
Declaration of Generative AI and AI-assisted technologies in the
writing process

During the preparation of this manuscript, the authors utilized Le
chat mistral AI and ChatGPT (OpenAI) to assist with language refine-
ment, grammatical corrections, and improvement of overall clarity. The
tool was not used for data generation, analysis, or interpretation of re-
sults. All scientific data, including study design, data analysis, and
conclusions, was solely the responsibility of the authors. The authors
thoroughly reviewed and revised the text to ensure its accuracy and
integrity, and they take full accountability for the final version of the
manuscript.

Declaration of Competing Interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Nadine Millot reports that financial support was provided by EIPHI
Graduate School (contract ANR-17-EURE-0002). Nadine Millot reports
that financial support was provided by FEDER-FSE Bourgogne. Nadine
Millot reports that financial support was provided by Cancéropôle Est
(project MangaColoRadio). Other authors declare that they have no
known competing financial interests or personal relationships that could
have appeared to influence the work reported in this paper.

Acknowledgments

The authors would like to express their sincere gratitude to Dr. Lucien Saviot for his valuable assistance with the Raman spectroscopy investigations, to Dr. Olivier Heintz for conducting the XPS analysis.

Mahran Bechetnia's mobility was supported by a scholarship pro-
vided by the Tunisian Ministry of Higher Education and Scientific
Research and EIPHI Graduate School (contract ANR-17-EURE-0002).

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.colsurfa.2026.139481.

Data availability

Data will be made available on request.

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