# Engineered biodegradables fibres for brain metal ion regulation

Tatiana Borisova, Juan Felipe Salazar Ariza, Natalia Pozdnyakova, Natalia Krisanova, Artem Pastukhov, Marina Dudarenko, Thomas Gréa, Alexandra Montembault, Imad Benkhaled, Noël Pinaud, et al.

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## ► To cite this version:

Tatiana Borisova, Juan Felipe Salazar Ariza, Natalia Pozdnyakova, Natalia Krisanova, Artem Pastukhov, et al.. Engineered biodegradables fibres for brain metal ion regulation. International Journal of Biological Macromolecules, 2025, 315, pp.144346. 10.1016/j.ijbiomac.2025.144346. hal-05110664

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Engineered biodegradables fibres for brain metal ion regulation

Tatiana Borisova<sup>a</sup>, Juan Felipe Salazar Ariza<sup>b,c</sup>, Natalia Pozdnyakova<sup>a</sup>, Natalia Krisanova<sup>a</sup>, Artem Pastukhov<sup>a</sup>, Marina Dudarenko<sup>a</sup>, Thomas Gréa<sup>b,c</sup>, Alexandra Montembault<sup>c</sup>, Imad Benkhaled<sup>d</sup>, Noël Pinaud<sup>d</sup>, Justine Deborne<sup>d</sup>, Yannick Crémillieux<sup>d</sup>, Philippe De Deurwaerdere<sup>e</sup>, Vladimir Lysenko<sup>b</sup>, Stéphane Palfi<sup>f</sup>, Olivier Tillement<sup>b</sup>, Laurent David<sup>c</sup>, Francois Lux<sup>b,g,*</sup>

ARTICLE INFO

Keywords:

Metallic homeostasis

Active hydrogel

Neurodegenerative disease

Chitosan Fibres

Biodegradable

A B S T R A C T

Exposure to environmental heavy metals and disruptions in physiological metal homeostasis are significant contributors to neurological and neurodegenerative disorders. To counteract heavy metal toxicity, a biopolymer consisting of chitosan functionalized with DOTAGA ligands (Chitosan@DOTAGA) was developed. By blending conventional chitosan with Chitosan@DOTAGA, biodegradable fibres with controlled diameters (192.3 ± 14.5 µm) were engineered and their mechanical properties characterized (Young's modulus of 0.115 ± 0.014 cN/dtex, yield stress of 0.288 ± 0.013 cN/dtex, tenacity of 0.47 ± 0.02 cN/dtex...). The chelation capacity of these fibres was assessed in an aqueous medium *via* ICP-MS analysis for various metal ions, including Cu(II), Cd(II), Pb(II), and Fe(III). Neuroprotective effects were evaluated *ex vivo* using nerve terminals isolated from rat brains exposed to Cu(II), Cd(II), Pb(II), and Hg(II); effectiveness was evidenced for all metals except Hg (II) testing transportation of excitatory and inhibitory neurotransmitters, L-[¹⁴C] glutamate (and [³H] GABA, and varied between 30 and 80 % within the line Cd(II)-Pb(II)-Cu(II). To validate the *in vivo* feasibility of the future use of Chitosan@DOTAGA for metal chelation in the central nervous system, Gd(III)-labeled fibres were implanted into rat brains for three months, with no observable inflammatory reactions detected *via* MRI or histology. Fibre biodegradability was assessed using MRI and ICP-MS analyses showing around 40 % of degradation after 17 weeks of implantation.

**Significance:** The ability to effectively remove excess metals from the brain is critical for treating various neurological disorders where metal accumulation contributes to disease pathology. Traditional chelation therapies face significant challenges, particularly in crossing the blood-brain barrier and selectively targeting affected areas. In this study, we introduce a biodegradable chelating polymer designed for implantation during surgical procedures. Our findings highlight neuroprotective properties *ex vivo* and promising safety *in vivo* in rodent model.

1. Introduction

Neurological disorders are the third leading cause of disability and

premature death in the European Union. Their prevalence and burden
increase, as highlighted in the recent report of Lancet Public Health
2020 [1]. Heavy metals such as lead, mercury and cadmium have been

<footnote><sup>a</sup> Palladin Institute of Biochemistry National Academy of Sciences of Ukraine, 9 Leontovicha Street, Kyiv 01054, Ukraine</footnote>

<footnote><sup>b</sup> Institut Lumière Matière UMR 5306 CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne, France</footnote>

<footnote><sup>c</sup> Université Claude Bernard Lyon 1, Institut National des Sciences Appliquées, Université Jean Monnet, CNRS, UMR 5223, Ingénierie des Matériaux Polymères (IMP), 15 bd Latarjet, 69622 Villeurbanne, France</footnote>

<footnote><sup>d</sup> Institut des Sciences Moléculaires, UMR 5255, CNRS, Université de Bordeaux, Bordeaux, France</footnote>

<footnote><sup>e</sup> Centre National de la Recherche Scientifique, Institut des Neurosciences Integratives et Cognitives d'Aquitaine, UMR, 5287 Bordeaux, France</footnote>

<footnote><sup>f</sup> Department of Neurosurgery, Henri Mondor Academic Hospital, Université Paris Est Créteil, INSERM Laboratory U955 of Translational NeuroPsychiatry, Institut Mondor de Recherche Biomédicale, France</footnote>

<footnote><sup>g</sup> Institut Universitaire de France (IUF), Paris, France</footnote>

<footnote>* Corresponding author at: Institut Lumière Matière UMR 5306 CNRS, Universite Claude Bernard Lyon 1, 69622 Villeurbanne, France.
E-mail address: francois.lux@univ-lyon1.fr (F. Lux).</footnote>
recognized as a major public health problem by the World Health Or-
ganization (WHO). Environmental pollution by these metals, along with
disruptions in the homeostasis of essential physiological metals, can
significantly contribute to the development of various neuropathologies
[2]. For example, copper plays a vitale role in the development and
function of the central nervous system (CNS) [3]. Disruptions in copper
metabolism, as seen in Menkes and Wilson's disease, lead to complex
neurodegenerative manifestations, underscoring the critical impact of
copper dishomeostasis [4]. Cadmium, a neurotoxic heavy metal, is
widely present in polluted environment, including water, air, food, and
smoke. Due to its low clearance, cadmium accumulates extensively in
the human body, with a half-life of 15–30 years. The CNS is particularly
vulnerable to cadmium toxicity, as the metal can exacerbate neurode-
generative diseases, contribute to peripheral polyneuropathies, and lead
to cognitive impairments in children [5]. Lead is a ubiquitous environ-
mental neurotoxic heavy metal that continues to pose a significant
global public health threat. Epidemiological studies have shown that
even low levels of lead exposure can negatively impact childhood IQ [6].
Iron, despite its essential role in multiple biological processes such as
oxygen transportation and enzymatic reactions [7], is also suspected to
play an important role in neurodegenerative diseases, where it appears
to promote protein aggregation and oxidative damage [8,9]. Notably,
studies have reported altered labile iron concentrations in the cerebro-
spinal fluid (CSF) of patients with mild cognitive impairment, with iron
levels of 0.46 ± 0.04 µM compared to 0.29 ± 0.04 µM in control patients
[10]. Moreover, several studies have associated iron accumulation with
tissue damage [11–13] as evidenced by the upregulation of ferritin and
heme oxygenase in the brain.

Novel drug targets for the abovementioned chronic disease are eagerly sought. While chelating agents have traditionally been used primarily for acute metal poisoning, their potential for preventive and prophylactic applications is highly promising [14]. Commonly used chelators include thiols such as Dimercaprol (British Anti-Lewisite, BAL), dimercaptosuccinic acid (DMSA or succimer), dimercaptopropane sulfonate (DMPS), penicillamine, CaNa₂EDTA. In some cases, a combination of these agents is recommended [14]. Wilson's disease is an example of a genetic disorder treated by chelation therapy. However, conventional chelation therapy is limited by secondary adverse effects, lack of compliance, and neurological worsening, as observed in some cases of Wilson's disease [15]. Furthermore, many chelating agents have limited access to the brain due to the presence of blood brain barrier, and their rapid elimination reduces their effectiveness in addressing gradual, chronic metal accumulation. Biodegradable chelating materials could offer a promising alternative if directly implanted within the affected area, particularly for neurodegenerative diseases such as Parkinson's disease, where redox-active metals like iron can contribute to disease progression [16]. This approach could be integrated during surgical procedures involving active implantable neurological devices, such as deep brain simulators [17].

Thanks to its attractive biological properties, including biocompati-
bility, biodegradability and antibacterial activity, chitosan is widely
recognized has a promising polymer for biomaterial development [18].
Chitosan is industrially derived from chitin which can be found in
crustacean shells, possibly as by-products of food industry [19]. It can be
processed into biodegradable hydrogels for various biomedical appli-
cations. These include wound healing and biomedical implants,
benefiting from its favorable properties such as biocompatibility, anti-
oxidant activity, and antibacterial effects [20–24]. Our team has
recently demonstrated the potential of functionalizing chitosan with
chelating molecules such as DOTAGA or DFO in combination with he-
modialysis, for the removal of copper or iron [25,26]. This approach has
led to two clinical trials currently recruiting for the treatment of Wilson's
disease (NCT05917327) and acute-on-chronic liver failure (ACLF)
(NCT06340269). Interestingly, Ficiara et al. developed nanobubble
formulations comprising glycol chitosan functionalized with DFO to
bind iron for neurological applications and have shown its impact on Aβ

misfolding [27]. Our team has also shown that chitosan coupled with
DFO or DOTAGA mixed with conventional chitosan forms chelating
hydrogels suitable for drug delivery [28,29] and that conventional
chitosan can be shaped into fibres whose dimensions and mechanical
properties can be finely tuned [30].

Integrating Chitosan@DOTAGA in the filaments could lead to
biodegradable fibres capable of removing metals from the implanted
zone. Previous work on implantable hydrogels has shown that mixing
Chitosan@DOTAGA and conventional chitosan lead to tunable biode-
gradability [31]. In this study, we demonstrate the feasibility of syn-
thesizing such fibres and evaluating their mechanical and chelating
properties on different metallic cations. One of the objectives of this
study was to assess the impact of these fibres on isolated rat cortex nerve
terinals (synaptosomes), i.e.: (i) evaluating the potential neurotoxic
properties of the fibre per se by analyzing their impact on key aspects of
glutamatergic and GABA-ergic neurotransmission; (ii) investigating the
ability of copper, mercury, cadmium and lead to influence extracellular
level of the excitatory neurotransmitter L-[¹⁴C]glutamate and the
inhibitory neurotransmitter [³H]GABA at the presynaptic site; (iii) demonstrating the ability of the fibres to mitigate heavy metal-induced
increase in the extracellular level of L-[¹⁴C]glutamate and [³H]GABA in
nerve terminals. In view of future applications of chelating fibres in
neuro-diseases, a preliminary in vivo proof of concept was also con-
ducted. The objectives were to evaluate the feasibility of implanting the
fibre in the rat brain, the relevance of using MRI to monitor the fibre in
the brain, and to obtain initial information on the biocompatibility of
the approach and of the biodegradability of the implanted filament. To
achieve this, the fibre was pre-labeled with gadolinium, enabling easy
MRI detection and ICP-MS quantification.

Taken together, this study encompasses the synthesis of a chelating
polymer and its derived fibres, the *in vitro* demonstration of their neu-
roprotective properties, and ultimately, the feasibility of their implan-
tation into the rodent brain with subsequent monitoring of their
biodegradability.

**2. Material and methods**

2.1. *Chitosan@DOTAGA synthesis*

2.1.1. *Chemicals*

Medical grade chitosan with low DA (6 % ± 0.5 %, determined by ¹H NMR spectroscopy using the Hirai method [32] (See Supporting Information Fig. S1) from Alaska snow crab was purchased from Matexcel (Bohemia, NY, USA, reference number NAT-0030, https://www.ma texcel.com/p/30/medical-grade-chitosan/). This chitosan was fully characterized. The weight-average and number-average molar masses (respectively M~w~ = 2.600 10⁵ g/mol and M~n~ = 1.300 10⁵ g/mol) were determined by size exclusion chromatography coupled with refractive index and multi-angle laser light scattering measurements at the chromatography platform of the Chemistry Institute of Lyon (ICL, https://www.iclyon.fr/). DOTAGA-anhydride (1,4,7,10-tetra-azacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid) was purchased in CheMatech (Dijon). GdCl₃, 6 H₂O (CAS 13450–84-5, 99.9 %), HEPES (≥99.5 % CAS 7365-45-9) Na₂SO₄ (ACS reagent, ≥99.0 %, anhydrous, powder, CAS 7757-82-6) NTA (ACS reagent, ≥99.0 % CAS 139–13-9), CuSO₄ (CuSO₄, 5 H₂O, ACS reagent, ≥98.0 %, CAS 7758-99-8), CdCl₂ (CdCl₂ × H₂O, CAS 654054–66-7, 99.995 %), Pb(NO₃)₂ (ACS reagent, ≥99.0 %, CAS 10099–74-8) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France), glacial acetic acid (AnalR NormaPur) grade was furnished by VWR (France). Standard Fe(III) solutions in 5 % HNO₃ were provided by SCP-Science (ICP Standard 1000 µg.mL, reference: 140–051-260).

**2.1.2. Functionalization of chitosan**

Functionalized chitosan (Chitosan@DOTAGA) was developed by
MexBrain, with its synthesis and characterization described in detail in a
previous study [28] (See Supporting Information Figs. S2 and S3, for the characterization of the functionalized chitosan). Briefly, the synthesis of the chelating polymer involved dissolving in a 50/50 (w/w) water-propanediol mixture, using acetic acid to protonate chitosan chains. DOTAGA-anhydride (1,4,7,10-tetra-azacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid) was then added to the solution to functionalize the polymer. The resulting product was purified by tangential filtration using a 100 kDa cut-off membrane. The amount of DOTAGA grafted onto the polymer was determined by UV-Vis titration in the presence of increasing concentration of Cu(II) following the copper chelation method described by Natuzzi et al, (See Supporting Information, Fig. S4) [26]. The Chitosan@DOTAGA polymer was found to have 0.654 mmol of DOTAGA grafted per gram of polymer, corresponding to a substitution degree of 17.0 ± 0.5 % (Refer to the supporting information Fig. S4 for details on the DOTAGA substitution degree).

### 2.1.3. Labelling of Chitosan@DOTAGA by Gd(III)

Gadolinium labelling of Chitosan@DOTAGA for in vivo experiments was carried out by adding 8.74 mL of a solution of 1 M gadolinium (prepared from GdCl₃, 6 H₂O) to 1 L of a Chitosan@DOTAGA solution at a concentration of 7 g.L⁻¹. The pH was then adjusted to 5.6 ± 0.1 using a 1 M NaOH solution, and the solution was stirred at 60 °C for 48 h. The concentration of chelated gadolinium was determined by inductively coupled plasma mass spectrometry (ICP-MS) to be 70.5 mg of Gd³⁺ per gram of polymer, corresponding to 70 % of the total DOTAGA sites. This concentration is sufficient for effective detection by MRI.

## 2.2. Fibre formation

### 2.2.1. Fibre synthesis

Chelating fibres were prepared by dispersing chitosan and Chitosan@DOTAGA in ultra-pure water at concentrations of 5 % and 10 % (w/w), respectively. Acetic acid was added to achieve stoichiometric protonation of the amine moieties in both polymers, and the mixture was mechanically stirred until a homogeneous solution was obtained. The precursor solution (dope) was then extruded following the same procedure described by Passieux et al. [30] (Fig. 1). Extrusion was performed at a linear velocity of $V_0 = 21 \text{ mm.s}^{-1}$ through a conical spinneret with a final diameter of 410 µm (Nordson EFD, smoothflow

tapered tips, 7,018,298), by applying pressure using a piston with a compressed air dispenser (NordsonOptimus I, NordsonEFD). The resulting extrudate was introduced into a 1 L bath of 3 M NaOH aqueous solution to induce gelation. A first motorized bobbin drove the fibre through two 5 L baths of ultrapure water for washing, while a second motorized bobbin directed it out of the washing baths and into the drying zone. Finally, a third motorized bobbin ensured the winding of the fibres. The speeds of the motorized bobbins were set at 21 mm.s⁻¹, 26 mm.s⁻¹, and 34 mm.s⁻¹, respectively.

Gadolinium-labeled fibres were prepared by dispersing three polymers, namely chitosan, Chitosan@DOTAGA and Chitosan@DOTAGA-Gd in ultrapure water at concentrations of 5 %, 8 %, 2 % (w/w), respectively. As before, acetic acid was added to achieve stoichiometric protonation of the amine moieties, ensuring polymer solubilization. The dope was then extruded following the same protocol used for the fabrication of the fibres without gadolinium. Thus, with this proportion of Chitosan@DOTAGA-Gd and its level of complexation, $0.70 \times 0.20 = 14\%$ of the total DOTAGA in the fibre is complexed with gadolinium.

The chelating fibres formulated above were used for in vivo experiments. The diameter of the dry fibres was determined after observation of the yarns with a Leica M205A stereomicroscope equipped with the Leica Application Suite v4.12 software, and image treatment using ImageJ. The titer (in dtex) was determined by weighting 2.5 m of fibre and converting it to 10,000 m.

### 2.2.2. Mechanical properties of the fibres

The tensile properties were assessed using a SHIMADZU AG-X plus device equipped with a 100 N load cell. The unidirectional tensile test was conducted at a constant speed of 10 mm/min for each sample at ambient temperature and relative humidity $Rh \sim 35\%$. The fibre was positioned between clamps establishing an effective initial length ($l_0 = 50 \text{ mm}$) and an initial deformation rate of 20 %/min. The tenacity ($T_e$) was calculated as the ratio of the applied force ($F_b$) at rupture to the yarn titer in dtex ($Y_t$) ($T_e = F_b/Y_t$); the nominal strain ($\epsilon$) was expressed as the ratio of the fibre's extension to the initial length ($\epsilon_N = \Delta l/l_0 = (l-l_0)/l_0$). The Young's modulus (E), tenacity ($T_e$), and strain-at-break ($\epsilon_b$) were determined from the stress-strain curves (see Figure Supporting Information Fig. 5), with five measurement replicates ($n = 5$). The same test was conducted for the fibres labeled with gadolinium, and the results were similar to those of the non-labeled fibres (Table 1).

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** **A:** Scheme of the lab-scale wet gel spinning setup for producing chitosan fibres out of the precursor solution containing chitosan and functionalized chitosan (chitosan@DOTAGA). It comprises a gelation bath (aqueous 3 M NaOH solution), three motorized bobbins that control the stretching rates, and an air gun for drying the fibres after they washing in two separate water baths. **B:** optical microscopy image of the fibre, with the left side showing the dry fibre (diameter ~ 180 µm) and the right side showing the rehydrated fibre in contact with a water drop (~900 µm).</figcaption></figure>
<caption>**Table 1**</caption>

Fibre mechanical characterization data obtained by uniaxial tensile test on 5 different samples of Chitosan 5%w/w Chitosan@DOTAGA 10% w/w fibre. The reported data are the mean of n = 5 tensile tests.

<table><thead><tr><th rowspan="2">Sample</th><th>Titer</th><th>Young's modulus<sup>a</sup></th><th>Yield stress<sup>a</sup></th><th>Yield strain</th><th>Tenacity<sup>a</sup></th><th>Strain at rupture</th><th>Diameter<sup>b</sup></th></tr><tr><th>(dtex)</th><th>(cN/dtex)</th><th>(cN/dtex)</th><th>(%)</th><th>(cN/dtex)</th><th>(%)</th><th>(µm)</th></tr></thead><tbody><tr><td>Chitosan 5% Chitosan@DOTAGA 10%</td><td>391.5</td><td>0.115 ± 0.014</td><td>0.288 ± 0.013</td><td>2.8 ± 0.2</td><td>0.47 ± 0.02</td><td>19.2 ± 2.2</td><td>192.3 ± 14.5</td></tr><tr><td>Chitosan 5% Chitosan@DOTAGA 8% Chitosan@DOTAGA-Gd 2%</td><td>295</td><td>0.151 ± 0.008</td><td>0.278 ± 0.018</td><td>2.0 ± 0.1</td><td>0.46 ± 0.02</td><td>18.7 ± 2.2</td><td>181.2 ± 21.8</td></tr></tbody></table>

$$d(\mu m) = \left( \frac{Yt(dtex) \times 100}{\frac{\pi}{4} \times \rho(g/cm^3)} \right)^{1/2}$$

### 2.2.3. Characterization of metal chelation by the fibres

Chelation experiments for each metal (Cu(II), Cd(II), Pb(II) and Fe (III)) were carried out separately. Each metallic solution was prepared in 20 mM HEPES at pH 7.4. To prevent lead precipitation, the pH of the lead solution was adjusted to 6.0. To prevent Fe(III) precipitation at pH 7.4, nitrotriacetic (NTA) chelator (Log Fe(III)-NTA: 15.9) [33] was added to the solution at a ratio of 1.1 NTA/1 Fe(III). As in previous works [28], Na₂SO₄ was also added at a concentration of 14 mM to prevent interactions between the NTA-Fe(III) complex and chitosan. The metal solutions were prepared by dissolving CuSO₄, CdCl₂, and Pb(NO₃)₂ at concentrations of 50 µM for Cu(II), 1 mM for Cd(II), and 2.5 mM for Pb (II) in the chelation baths. The iron solution was prepared by diluting a ferric solution (1000 µg.mL⁻¹ of Fe(III) in 5 % HNO₃) up to desired concentration of 0.47 µM. Cu(II), Pb(II), and Cd(II) concentrations correspond to the metal levels at which significant extracellular neurotransmitter levels (indicating neurotoxicity) were measured in the synaptosome solution. The Fe(III) concentration was chosen based on literature values of labile iron in Alzheimer's patients with cognitive impairment [34]. These concentrations were chosen to be close to those used in biological experiments that have determined neuronal toxicities for the different metals. Subsequently, 5 cm of fibre were immersed into 10 ml of each metallic solution (each experiment was conducted in triplicate). The samples were stirred at room temperature for one week. The amount of metals extracted by the fibre was determined using inductively coupled plasma spectrometry (ICP-MS) after the fibres were mineralized by microwave treatment with 3 ml of HNO₃ (69 %). The analysis was performed using a Perkin Elmer NexION2000 equipped with Syngistix software (Perkin Elmer, Version 2.3) and ESI SC-FAST sample introduction in standard mode. The samples were prepared by dilution in an aqueous solution of 1 % HNO₃ (v/v). Samples from the supernatant were also collected at different time points and analyzed in the same manner as the mineralized fibres.

## 2.3. Ex vivo experiments

### 2.3.1. Ethical approval

Animals (Wistar rats, males, body weight of appx 120 g) were kept in the animal facilities of the Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, housed in a temperature-controlled room (22–23 °C), and provided *ad libitum* with water and standard diet using dry food pellets. The animal experiments were performed in accordance with the Guidelines of the European Community (2010/63/ EU) and local laws and policies, and were approved by the Animal Care and Use Committee of the Palladin Institute (Protocol from September 21, 2020). All animal studies were reported in accordance to the ARRIVE guidelines for reporting experiments involving animals [35,36]. The

total number of rats used in the study was 48. Specifically, 48 animals were used for measuring the extracellular levels of L-[¹⁴C] glutamate and [³H] GABA in nerve terminals while 16 rats were shared for fluorimetry.

### 2.3.2. Materials

EGTA, EDTA, HEPES, Ficoll 400, Sigma-Fluor® High Performance LSC Cocktail, the analytical grade salts were purchased from Sigma (USA); L-[¹⁴C] glutamate and [³H] GABA (γ-[2,3-3H(N)]-aminobutyric acid) were from Perkin Elmer (Waltham, MA, USA). Rhodamine 6G were obtained from Molecular Probes (USA).

### 2.3.3. Statistical analysis

The experimental data were expressed as the mean ± S.E.M. of n independent experiments. The difference between two groups was compared by one-way ANOVA. Differences were considered significant, when *p* < 0.05.

### 2.3.4. Isolation of nerve terminals (synaptosomes) from the cortex regions of the rat brains

The cortex brain region isolated from decapitated rats was rapidly removed, and then homogenized in the ice-cold solution containing 0.32 M sucrose, 5 mM HEPES-NaOH, pH 7.4, and 0.2 mM EDTA. Each synaptosome preparation was obtained from one rat, and each measurement was done in triplicate. The synaptosome preparations were obtained using differential and Ficoll-400 density gradient centrifugations of rat brain homogenate according to previous works [37]. The synaptosome preparations were used in the experiments during 2–4 h after isolation. The standard salt solution contained (in mM): NaCl 126; KCl 5; MgCl₂ 2.0; NaH₂PO₄ 1.0; HEPES 20, pH 7.4; and D-glucose 10. Total protein concentration in synaptosome preparation was examined according to Larson et al. [38].

### 2.3.5. The extracellular level of L-[¹⁴C] glutamate in the nerve terminal preparations

The synaptosome preparations were diluted in the standard salt solution to reach a concentration of 2 mg of total protein/ml, and after pre-incubation at 37 °C for 10 min were loaded with L-[¹⁴C] glutamate (2.81 µM, 1 µCi/ml) in the standard salt solution at 37 °C for 10 min. After such loading procedure, the synaptosome suspensions were washed with 10 volumes of ice-cold standard salt solution and centrifuged; the pellets were re-suspended in the standard salt solution to reach a final concentration of 1 mg of total protein/ml. Synaptosome suspensions (125 µl; 0.5 mg of total protein/ml) were pre-incubated at 37 °C for 10 min; then pieces of the chitosan fibre were added and incubated with synaptosomes for 10 min, and then sedimented using a

<footnote>a The values of stress expressed in textile units cN/dTex can be converted in MPa using the estimated density of ρ ~ 1.3 g/cm³ according to: σ(MPa) = σ(cN/dtex) ρ(g/cm³)*100.</footnote>

<footnote>b Values deduced from optical microscopy measurements. The apparent diameter d of the fibre can be deduced from the titer Yt(dtex) with greater precision using:</footnote>
microcentrifuge (20 s at 10000 g). The extracellular level of L-[¹⁴C] glutamate was recorded in the aliquots of supernatants (100 µl) and pellets using liquid scintillation counting with Sigma-Fluor® High Performance LSC Cocktail (1.5 ml), and the values were expressed as the percentage of total accumulated synaptosome L-[¹⁴C] glutamate [39,40]. L-[¹⁴C] glutamate release data were collected in triplicate from several independent experiments performed with different synaptosome preparations.

### 2.3.6. The extracellular level of [³H] GABA in the nerve terminal preparations

The synaptosome preparations were diluted in the standard salt solution up to 2 mg of total protein/ml, and after their pre-incubation at 37 °C for 10 min were loaded with [³H] GABA (50 nM, 4.7 µCi/ml) in the standard salt solution for 10 min. GABA transaminase inhibitor amino-oxyacetic acid at a concentration of 100 µM was used in the experiments of [³H] GABA loading and release to minimize the formation of GABA metabolites. After loading, the synaptosome suspensions were washed with 10 volumes of ice-cold standard salt solution. The pellets were resuspended in the standard salt solution to reach a protein concentration of 1 mg of total protein/ml. Synaptosome suspensions (120 µl) were pre-incubated at 37 °C for 10 min, then the pieces of the chitosan fibres were added and incubated for 10 min, and sedimented using a micro-centrifuge (20 s at 10,000 g). [³H] GABA radioactivity was measured in the aliquots of supernatants (90 µl) by liquid scintillation counting with Sigma-Fluor® High Performance LSC Cocktail (1.5 ml), and the values were expressed as the percentage of total accumulated synaptosome [³H] GABA [41]. [³H] GABA release data were collected in triplicate from several independent experiments performed with different synaptosome preparations.

### 2.3.7. The plasma membrane potential ($E_m$) in nerve terminals

The membrane potential was measured using a potentiometric fluorescent dye rhodamine 6G (0.5 µM) based on its potential-dependent binding to the plasma membrane [40,42,43]. After preincubation at 37 °C for 10 min, the suspension of synaptosomes (0.2 mg of total protein/ml) was added to stirred thermostated cuvette. In order to estimate changes in the plasma membrane potential the ratio ($F$) as an index of membrane potential was calculated according to: $F = F_t/F_0$, where $F_0$ and $F_t$ are fluorescence intensities of a fluorescent dye in the absence and presence of the synaptosomes, respectively. $F_0$ was calculated by extrapolation of exponential decay function to $t = 0$. Fluorescence measurements with rhodamine 6G were carried using a Hitachi 650-10 S spectrofluorimeter or a QuantaMasterTM 40 (PTI, Inc., Canada) spectrofluorimeter at 528 nm (excitation) and 551 nm (emission) wavelengths (slit bands 5 nm each).

## 2.4. *In vivo* feasibility study

### 2.4.1. Animals

Three female rats of Wistar strain (9 weeks old, 190 g) were used for *in vivo* experiments. Animals were procured from Janvier Laboratory (Le Genest-Saint-Isle, France). They were kept in standard housing conditions (12 h light-dark cycles) with a standard rodent chow and water available *ad libitum*.

### 2.4.2. Ethical approval

All animal procedures were performed in accordance with the rules of the European Committee Council Directive 2010/63/ EU after validation by our local ethical committee and authorization from the French Ministry of Research (University of Bordeaux, reference number 39329-2022033111368258).

### 2.4.3. Implantation of the fibre in the brain of rodents

During surgery, rats were anesthetized with 2.5 % isoflurane in a mixture of air/O₂ (70/30) *via* a facial mask and fixed in a stereotaxic

frame. After incision of the skull skin, a small hole was drilled in the skull using a 1 mm diameter drill bit for the insertion of the Gd-labeled chitosan chelating fibre. The hole was positioned +3 mm medio/lateral right hemisphere relative to bregma according to the atlas of the rat brain [44] for the implantation of the fibre in the right striatum. The device used for the implantation of the chelating fibre is shown in Fig. 2. It consists of a polyimide cannula (with inner/outer diameter 190 µm/ 220 µm) glued to a support of sufficient size to be held by a stereotaxic device clamp. Once the cannula positioned in the center of the hole drilled in skull, the chelating fibre (3 mm long and ~ 180-µm diameter) was introduced into the cannula and pushed into the brain with a microwire (150-µm diameter) of insulated copper. Once the fibre positioned in the striatum, the cannula and copper wire were removed and the skin of the animal was stitched up.

Sterile surgical procedures were used for the implantation of the fibre, including moist heat sterilization of the cannula, copper wire and fibre itself. After surgery, the rats were housed individually and received doses of buprenorphine 0.05 mg/kg every 12 h for 48 h. The three animals were then housed in the same cage. The weight of the animals was controlled every week.

### 2.4.4. MRI following the degradation of the Gd-labeled chitosan fibre implanted in the brain

MRI experiments were performed on a preclinical 4.7-T Bruker BioSpec 47/20 MRI (Bruker BioSpin, Ettlingen, Germany) running under Paravision 6.0.1. A quadrature birdcage coil with inner diameter of 86 mm was used for RF excitation and a phased array surface coil dedicated to rat head for RF detection (Bruker, Ettlingen, Germany). One implanted animal was imaged at day 0, 3, 7 and then once a week up to 8 weeks. The follow-up MRI protocol included three MRI acquisitions with $T_1$ weighting (FLASH sequence, TE 3.5 ms, TR 200 ms, flip angle 60°, slice thickness 0.5 mm, in plane resolution 0.1 × 0.1 mm), $T_2$ weighting (TSE sequence, TE 36 ms, TR 2200 ms, slice thickness 0.5 mm, in plane resolution 0.14 × 0.14 mm) and $T_2^*$ parametric imaging (MGE sequence, 12 echoes, TE 2.2 ms, TR 200 ms, slice thickness 0.5 mm, in plane resolution 0.14 × 0.14 mm). At each imaging session, the diameter and length of the implanted fibre was measured on the $T_1$-weighted images. The SNR (signal-to-noise ratio) were measured in two regions of interest (ROIs) corresponding to the fibre and to brain tissue adjacent to the fibre. The $T_2^*$ values (mean and standard deviation) were measured as well in these two ROIs. The CNRs (contrast-to-noise ratio) for $T_1$ and $T_2$ weighted acquisitions were computed as the difference between the SNRs of the fibre and brain tissue.

### 2.4.5. Histology of brain after sacrifice of the animals

All animals were euthanized past 3 months post implantation of the Gd-labeled chitosan fibre and their brain were collected for later analysis. In order to determine the amount of Gd in the brain and estimate the degradation of the fibre, one rat brain was divided into two hemispheres (left hemisphere and right hemisphere containing the fibre). Each hemisphere was mineralized using microwave treatment with 3 ml of HNO₃ (69 %). The samples were prepared by dilution in an aqueous solution of 1 % HNO₃ (v/v) and analyzed by ICP-MS (samples were prepared and analyzed in triplicates). In order to confirm resorption through the evolution of the size of the fibre and verify the integrity of the tissue, another brain was frozen and processed for histological staining. The forebrain was cut at 60 µm on a cryostat (−20 °C; CM1800, Leica, Paris France) in the coronal plane. Slices were directly placed on slides and stained with cresyl violet after progressive dehydration in baths of ascending concentration of alcohol. The striatum was observed under light microscope to evaluate the presence of gliosis surrounding the fibre and calculate the diameter of the fibre.
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Photograph of the chelating fibre insertion device, in the centre of the figure, with enlarged views of the cannula, fibre and copper microwire before and after insertion of the fibre. On the right side of the figure, the insertion procedure before implantation (1), after insertion of the cannula (2), after pushing the copper microwire (3), and after removal of the cannula from the brain (4).</figcaption></figure>

## 3. Results

### 3.1. Formation of fibres and characterization of their chelating capacities

Chitosan@DOTAGA was obtained by direct functionalization of a low DA chitosan by DOTAGA anhydride in hydro-alcoholic media. The grafting ratio of 17.0 ± 0.5 % was the higher ratio obtained by varying DOTAGA anhydride amounts in the reaction medium (data not shown). It was possible to obtain fibres from a dope consisting in a mixture of chitosan and Chitosan@DOTAGA with tunable diameters (data not shown) by varying conical spinneret diameters and velocities of the

motorized bobins on a lab-scale homemade apparatus (Fig. 1). As previously highlighted, Cu(II), Cd(II), Pb(II) and Fe(III) are known to be neurotoxic. To assess the chelation efficacy of the fibre for these distinct metals, the fibre underwent exposure to ~50 µM of CuSO₄, ~1 mM of CdCl₂, ~2.5 mM of Pb(NO₃)₂, and 0.47 µM of Fe(III) in aqueous media, as illustrated in Fig. 3A. Concentrations of metals have been chosen in order to be in line with the relevant concentrations used in the biological experiments.

Cd(II) (11.86 ± 0.34 µmol) and Pb(II) (23.08 ± 0.24 µmol) are in large excess compared to the calculated amount of DOTAGA in 5 cm of fibre (0.64 µmol). However, in the case of the Cu(II) solution (0.36 ±

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** A representation of the chelation of metals by 5 cm of fibre in a metallic solution (created with BioRender.com). Evolution B of Cu(II) (CuSO₄ 50 µM), C Cd(II) (CdCl₂ 1 mM), D of Pb(II) (Pb(NO₃)₂ 2.5 mM) and E of iron (III) (Fe-NTA 0.5 µM) concentrations over time in the presence of 5 cm of fibre in 10 ml of HEPES 20 mM pH 7.4 determined by ICP-MS. The solution containing Pb(II) was at a lower pH (5.5) to avoid its precipitation.</figcaption></figure>
0.03 µmol), there is a slight excess of DOTAGA and in the case of the Fe (III) solution, the DOTAGA present in the fibres is in large excess compared to the Fe(III) (4.7 ± 0.3 nmol) in solution. Consequently, the ratios of M<sup>n+</sup>/DOTAGA varied to 18.6, 36, 0.6 and 0.007 for cadmium, lead, copper, and iron respectively. DOTA exhibits a strong complexation constant for the different studied metals as shown by the different complexation constants: Logβ<sub>Pb II</sub> = 22.69 [45], Logβ<sub>Cd II</sub> = 21.3 [46], Logβ<sub>Cu II</sub> = 22.44 [45] and Logβ<sub>Fe III</sub> = 29.4 [45].

By monitoring the metal concentrations in the supernatant over time using ICP-MS, it becomes apparent that the fibre extracts Cu(II), Cd(II), Pb(II) and Fe(III) from the solutions. Furthermore, the extraction equilibrium was achieved after 2 days for Cd(II) and Pb(II) (Fig. 2B, D), whereas for Cu(II) (Fig. 2C), equilibrium was reached no earlier than the fifth day. The metallic extraction of the fibre was calculated from the ICP-MS analysis of the fibres by the following Eq. 1:

$$ \text{Metallic extraction} = M^{n+}\text{fiber}_f - M^{n+}\text{fiber}_i \quad (1) $$

where M<sup>n+</sup> fibre<sub>f</sub> corresponds to the amount of metallic ion in the fibre at the end of the experiment, M<sup>n+</sup> fibre<sub>i</sub> the amount of metallic ion measured in the control fibre unexposed to metallic solution. The metallic extraction by the 5 cm fibres is as follows: 2.88 ± 0.04 µmol for Cd(II), 1.80 ± 0.05 µmol for Pb(II), 0.25 ± 0.01 µmol for Cu(II) and 3.3 ± 0.2 nmol for Fe(III). Which represents 24.3 ± 0.4 %, 7.8 ± 0.2 %, 65.3 ± 0.5 %, and 70.7 ± 5.1 % of extracted Cd(II), Pb(II), Cu(II) and Fe(III) respectively. The amount of metals extracted for cadmium and lead exceeds the amount of DOTAGA present in the fibre (0.64 µmol). This suggests that not all metallic cations are chelated by DOTAGA, and some of the metals interact with chitosan. In fact, chitosan is well known to possess chelating properties [47], which are dependent on the nature of the cation as well as other species surrounding the cation, such as its counterion. This problem was previously addressed in the iron chelation experiment by introducing Na<sub>2</sub>SO<sub>4</sub> [29], which is known to interact with chitosan by complexing with the amines moieties [48,49]. This interaction prevents the NTA-Fe complex from interacting with chitosan. Therefore, in this experiment, Fe(III) is effectively chelated by the DOTAGA present within the fibre. To account for the effect of the counterion on chitosan's chelation properties, future work should be conducted under conditions where the same counterion is used. No mercury chelation experiment was made due to the weak affinity of the DOTA chelator towards Hg(II) as shown by Randhawa et al. [50], although chitosan was reported to exhibit some mercury absorption capacity [51].

## 3.2. Assessment of neuro compatibility of the fibres using brain nerve terminals

The extracellular level of neurotransmitters in the nerve terminal preparations represents a dynamic balance of transporter-mediated uptake and release as well as unstimulated leakage of these neurotransmitters. This parameter is very sensitive to energy status of nerve terminals and plasma membrane integrity [52,53].

In the first series of the experiments, the effects of the fibres together with its constituent DOTAGA on the extracellular level of neurotransmitters L-[¹⁴C] glutamate and [³H] GABA were assessed in the nerve terminal preparations. Two fibres lengths were analyzed (based on drafting DOTAGA chelating capability), i.e. 0.5 cm and 5 cm (with a fibre diameter close to 192 µm, relevant for future in vivo application). It was shown that the fibres did not change the extracellular level of neither L-[¹⁴C] glutamate nor [³H] GABA (Table 2A). Whereas, DOTAGA starting from a concentration of 1.25 mM increased the extracellular level of L-[¹⁴C] glutamate and [³H] GABA in nerve terminal preparations (Table 2B).

The membrane potential was monitored using the potential-sensitive

<caption>**Table 2**</caption>

The extracellular level of excitatory and inhibitory neurotransmitters L-[¹⁴C] glutamate and [³H] GABA, respectively, in the nerve terminal preparations in the presence of the fibre (A) and its constituent DOTAGA (B). Data are the mean ± SEM. ***, *p* < 0.001; n.s., no significant differences as compared to the appropriate control.

<table><thead><tr><th colspan="3">A</th></tr><tr><th>Samples</th><th>The extracellular level of L-[¹⁴C] glutamate in the nerve terminal preparations, % of total accumulated label</th><th>The extracellular level of [³H] GABA in the nerve terminal preparations, % of total accumulated label</th></tr></thead><tbody><tr><td>Control</td><td>17.91 ± 0.59 %</td><td>18.84 ± 0.51 %</td></tr><tr><td>Chitosan 5 %</td><td>17.46 ± 0.53 %; n.s.</td><td>18.53 ± 0.42 %; n.s.</td></tr><tr><td>Chitosan@DOTAGA 10 %</td><td>[F<sub>(1,94)</sub> = 0.34; p = 0.55; n = 48]</td><td>[F<sub>(1,94)</sub> = 0.22; p = 0.63; n = 48]</td></tr><tr><td>Fibre length: 0.5 cm</td><td>18.37 ± 0.62 %; n.s.</td><td>18.41 ± 0.43 %; n.s.</td></tr><tr><td>Chitosan 5 %</td><td>[F<sub>(1,94)</sub> = 0.28; p = 0.59; n = 48]</td><td>[F<sub>(1,94)</sub> = 0.42; p = 0.51; n = 48]</td></tr><tr><td>Chitosan@DOTAGA 10 %</td><td></td><td></td></tr><tr><td>Fibre size 5 cm</td><td></td><td></td></tr><tr><td colspan="3">B</td></tr><tr><td>Control</td><td>19.34 ± 0.46 %</td><td>17.11 ± 0.54 %</td></tr><tr><td>DOTAGA (0.5 mM)</td><td>18.65 ± 0.73 %; n.s.</td><td>17.64 ± 0.67 %; n.s.</td></tr><tr><td></td><td>[F<sub>(1,60)</sub> = 0.67; p = 0.41; n = 16]</td><td>[F<sub>(1,60)</sub> = 0.39; p = 0.53; n = 16]</td></tr><tr><td>DOTAGA (1.25 mM)</td><td>29.90 ± 1.17 % ***</td><td>26.02 ± 0.71 %***</td></tr><tr><td></td><td>[F<sub>(1,60)</sub> = 75.32; p < 0.001; n = 16]</td><td>[F<sub>(1,60)</sub> = 106.81 p < 0.001; n = 16]</td></tr><tr><td>DOTAGA (5 mM)</td><td>90.25 ± 1.13 % ***</td><td>91.59 ± 1.27 %***</td></tr><tr><td></td><td>[F<sub>(1,60)</sub> = 3582.97; p < 0.001; n = 16]</td><td>[F<sub>(1,60)</sub> = 3103.76 p < 0.001; n = 16]</td></tr></tbody></table>

fluorescent dye rhodamine 6G. F<sub>st</sub>, the membrane potential index at the steady state level, was first achieved after 5 min, and it was set as 100 % in statistical calculations. In such fluorometric experiments, shown in Fig. 4 A and B, it can be concluded that the fibres did not change the membrane potential of nerve terminals and, therefore, so did not depolarize their plasma membrane. On the contrary, DOTAGA molecule starting from a concentration of 1.25 mM depolarized the plasma membrane of nerve terminals (Fig. 4B).

Consequently, the fibres did not have any effects on the extracellular level of the neurotransmitters and the membrane potential of nerve terminals, whereas its constituent DOTAGA starting from a concentration of 1.25 mM increased this level and depolarized the plasma membrane of nerve terminals. Therefore, the grafted macromolecular structure of chitosan@DOTAGA and the fibre microstructure overcome the neurotoxic features of its exhibited by free DOTAGA.

## 3.3. Capability of the chitosan 5 % Chitosan@DOTAGA 10 % fibre to mitigate the extracellular level of L-[¹⁴C] glutamate induced by copper, mercury, cadmium, and lead in nerve terminal preparations

All experimental data were analyzed by two-way ANOVA followed by Tukey's post hoc test using the fibre and CuSO<sub>4</sub>/HgCl<sub>2</sub>/CdCl<sub>2</sub>/PbAc presence as the independent factors. As shown in Fig. 5, different concentrations of metals are necessary to induce significant increase of the extracellular level of L-[¹⁴C] glutamate in nerve terminal preparations ranging from 8 µM for HgCl<sub>2</sub> to 2.5 mM for PbAc. Interestingly, these concentrations are in the µM range for HgCl<sub>2</sub> and CuSO<sub>4</sub> and in the mM range for CdCl<sub>2</sub> and PbAc. The fibre mitigated Cu(II) (Fig. 5A), Cd(II) (Fig. 5C) and Pb(II) (Fig. 5D)-induced increase in the extracellular level of L-[¹⁴C] glutamate in nerve terminal preparation in a dose-dependent manner, contrary to Hg(II) where no significant change is observed for the two fibres (Fig. 5B) (All the values can be fund in Table S1).
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Fluorescence experiments: The membrane potential of nerve terminals in the presence of the fibre (0.5–5 cm) (A, B) and DOTAGA alone (B). A – The suspensions of synaptosomes were equilibrated with potential-sensitive dye rhodamine 6G (0.5 mM). When the steady level of the dye fluorescence had been reached, the fibre was added to synaptosomes (marked by arrows). Traces are typical and represent 16 experiments performed with different synaptosome preparations. B – An increase in the fluorescence signal of rhodamine 6G in response to application of water (control), the Chitosan 5 % Chitosan@DOTAGA 10 % fibres (0.5–5 cm), DOTAGA (0.5–5 mM). Data are the mean ± SEM. ***, p < 0.001 as compared to the control; n.s., no significant differences; n = 16.</figcaption></figure>

**3.4. Capability of the chitosan 5 % Chitosan@DOTAGA 10 % fibre to mitigate changes in the extracellular level of [³H] GABA induced by copper, mercury, cadmium, and lead in nerve terminal preparations**

All experimental data were also analyzed by two-way ANOVA followed by Tukey's *post hoc* test using the fibre and CuSO₄/HgCl₂/CdCl₂/PbAc as the independent factors in these experiments. Interestingly, the same concentrations of metallic cations also induce significant increase of the extracellular level of [³H] GABA in nerve terminal preparation (Fig. 6). Similarly, a mitigation of the effect of the metals is also observed in a dose dependent manner for Cu(II) (Fig. 6A), Cd(II) (Fig. 6C) and Pb(II) (Fig. 6D) while no mitigation is observed for Hg(II) (Fig. 6B) (All the values can be found in Table S1).

In view of potential translation, feasibility studies of fibre implantation in the brain were performed and monitored via MRI, using gadolinium-labeled Chitosan@DOTAGA as a contrast agent.

**3.5. Assessment of biocompatibility of the fibre and biodegradability**

**3.5.1. In vivo studies and MRI following of the biodegradability**

If one except the first day following the surgery, the animals did not show signs of physical suffering, discomfort and abnormal behavior

during the three months post-fibre implantation. Their weight increased in average from 190 to 300 g during these three months.

A selection of MRI images acquired post implantation of the fibre are shown in Fig. 7.A-D3. A slice of a T₁-weighted MRI coronal acquisition acquired at D0 is shown in Fig. 7 A; the white spot pointed by the red arrow corresponding to the implanted fibre. T₁-weighted sagittal images featuring the fibre acquired at D0, D21 and D49 are presented in Fig. 7 B1-3. The corresponding T₂-weighted images and T₂* maps are shown respectively in Fig. 7 C1-3 and D1-3.

After 3 h of implantation, the length of the implanted Chitosan 5 % Chitosan@DOTAGA 8 % Chitosan@DOTAGA-Gd 2 % fibre, as assessed on sagittal T₁-weighted images, were measured to be close to the initial length before implantation (i.e. L = 3 mm). However, the diameter of the fibre increased from 180 µm before implantation to a value ranging between 300 and 400 µm due to high (anisotropic) swelling properties of the material in aqueous medium as illustrated in Fig. 1.B. The length and diameter of the implanted fibre did not exhibit significant changes during the first 7 weeks of follow-up. However, after the 13th week, a notable difference was observed when measuring the fibre section, i.e. the fibre area in the transverse plane. We observed a decrease in the fibre area from 1.15 ± 0.29 mm² after 3 h of implantation to 0.69 ± 0.23 mm² at the conclusion of the experiment (17th week). This corresponds
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** The extracellular level of L-[¹⁴C]glutamate in the nerve terminal preparations in the control and in the presence of the fibre (0.5 and 5 cm) and CuSO₄ (50 µM) (A); HgCl₂ (8 µM) (B); CdCl₂ (1 mM) (C); PbAc (2.5 mM) (D). Data are the mean ± SEM. ***, *p* < 0.001; **, *p* < 0.01 as compared to the appropriate control; ###, *p* < 0.001; ##, *p* < 0.01 as compared to the appropriate metal; n.s., no significant differences; *n* = 16.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** The extracellular level of [³H]GABA in the nerve terminal preparations in the control and in the presence of the fibre (0.5 and 5 cm) and CuSO₄ (50 µM) (A); HgCl₂ (5 µM) (B); CdCl₂ (1 mM) (C); PbAc (1 mM) (D). Data are the mean ± SEM. ***, *p* < 0.001; *, *p* < 0.05 as compared to the appropriate control; ###, *p* < 0.001; #, *p* < 0.05 as compared to the appropriate metal; n.s., no significant differences; *n* = 16.</figcaption></figure>

to a 40.6 ± 5.1 % degradation of the fibre (Fig. 7 E-F) assessing the biodegradable properties of the implanted materials. Conversely, ICP-MS analysis of one of the brains reveals that 46 % of the total Gd is undetected within the fibre's zone, confirming that approximately half of the fibre has degraded after 17 weeks of implantation in the rat's brain.

The Contrast to Noise Ratio (CNR) between the fibre and the adjacent brain tissue for the T₁-weighted acquisition ranged from 5.5 to 11 (initial value equal to 8 and typical standard deviation value equal to 2). These positive values reflect the hyperintense MRI intensities in the fibre, presumably due to the presence of the chelated gadolinium ion acting as positive contrast agent, permanently chelated with DOTAGA
<figure><img src="image_10.png" /><figcaption>Fig. 7. A-D) MRI slices including the chelating fibre acquired with different MRI contrast (T₁ and T₂ weighting and quantitative T₂* map) at three time points (D0, D21 and D49). E) Variation of the area of the fibre measured by MRI over time in the transverse plane. F) Degradation of the fibre depicted by the decrease of the fibre area over time, as determined from MRI images (transverse plane).</figcaption></figure>

grafts.

The CNR between the fibre and the adjacent brain tissue for the T₂-weighted acquisition ranged from –5.4 to –9.5 (similar initial and final values and typical standard deviation value equal to 3). These negative values reflect the hypointense MRI intensities in the fibre, again presumably due to the presence of the chelated gadolinium ion acting as negative contrast agent and to numerous labile relaxing protons from hydroxyl and amine groups in the chitosan polymer.

The T₂* values measured in the fibre ranged from 16 ms to 19.7 ms with typical standard deviation equal to 5 ms. These values were lower to those obtained in adjacent brain tissue (35 to 51 ms) and are probably related to the presence of the chelated gadolinium ions. No trend in the temporal evolution of CNR and T₂* values could be evidenced with certainty. The corresponding graphs are shown in Fig. 8.

### 3.5.2. Histology after sacrifice of the rodents

The histological analysis confirmed the presence of the fibre in the striatum (Fig. 9A). It was observed on three adjacent cuts of 60 µm width. Correspondingly, its thickness was calculated at higher magnification and was close to 130 µm (Fig. 9B), evidencing again bioresorption with a decrease in diameter in comparison with the initial fibres (182 µm). The histological analysis of these slices revealed absence of gliosis in the tissue surrounding the fibre (Fig. 9A/B). Of note, MR images taken a few hours after fibre implantation on D0 showed no signs of hemorrhage or bleeding. T₁ and T₂ images also showed no signs of acute or chronic inflammation after 49 days of implantation. These observations are in agreement with those made on histological sections including the fibre.

<figure><img src="image_11.png" /><figcaption>Fig. 8. Follow-up graphs of T₁ and T₂-weighted CNR (left plots) and T₂* values in the fibre and in brain. Error bars correspond to the standard deviation of the measurements.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Histological analysis of the fibre in the striatum at low (A) or high (B) magnification. The yellow line corresponds to the thickness of the fibre (130 µm). No sign of gliosis was observed at low and high magnification. cc, corpus callosum; ac, anterior commissure.</figcaption></figure>

4. Discussion

Environmental pollution from metal overload and endogenous metal
dysregulation are increasingly recognized as key contributors to the
etiology of neurological diseases, such as Wilson disease, neuro-
degeneration with brain iron accumulation, Alzheimer's disease, Par-
kinson's disease [54]. The majority of research efforts has focused on the
development of chelators aimed at remediating heavy metal contami-
nation in the environment [55]. We report here the safety and efficacy of
fibres composed of metal chelator-grafted chitosan, assessed both *ex vivo*
on rat brain synaptosomes and *in vivo* in the rat striatum. Our data show
that the fibres mitigate the effects of most heavy metals application on
both glutamate and GABA extracellular levels. Additionally, the fibres
remained stable in the brain for several weeks without signs of tissue
damage and exhibited quantifiable biodegradability. The goal of these
experimental developments is to further implant the fibre in specific
brain regions affected by neurological diseases, where heavy metal
accumulation is believed to be toxic.

In this study, we demonstrated that copper, mercury, cadmium, and
lead increased the extracellular level of L-[¹⁴C]glutamate and [³H]GABA
in nerve terminals with varying potency and efficiency (Figs. 5 and 6). In
particular, the calculated effectiveness of the fibre to mitigate neuro-
toxic effects of heavy metals was 80 and 76 % for Cu(II) in L-[¹⁴C]
glutamate and [³H]GABA experiments, respectively, 58 % for Pb(II) and
both neurotransmitters, and 27 and 45 % for Cd(II), respectively.
Notably, the addition of 50 µM Cu(II) to the nerve terminals resulted in a
two-fold increase in the extracellular levels of L-[¹⁴C]glutamate with an
even greater increase in the extracellular levels of [³H]GABA. A con-
centration of 8 µM Hg(II) was sufficient to increase the extracellular
levels of both neurotransmitters, with similar efficacy to Cu(II) for
glutamate, but weaker for GABA. The effects of Cd(II) application (1

mM) were comparable for both neurotransmitters, with levels approxi-
mately doubled, while lead (2.5 mM) had a slightly lower efficacy,
though it affected both neurotransmitters similarly. The distinct effects
of these metals in terms of potency and/or efficacy towards gluta-
matergic versus GABAergic nerve terminals suggested the involvement
of different mechanisms triggered by heavy metals in these terminals.
The mechanisms are likely complex, as the measured neurotransmitters
are related to a dynamic balance of neurotransmitter uptake and tonic
unstimulated release [52,53]. The literature data on molecular mecha-
nisms of heavy metal neurotoxicity underlined the influence of heavy
metals on synaptic structure and function, metal-induced production of
reactive oxygen species, targeting sulfhydryl groups of proteins involved
in synaptic functioning that in turn can explain our results on metal-
induced changes in the extracellular level of L-[¹⁴C]glutamate and
[³H]GABA in nerve terminals [56]. The reported increase is therefore
not necessarily related to an increase in the Ca(II)-dependent, vesicular
exocytosis as evidenced with Cd(II) which is a non-selective blocker of
Ca(II) channels [57].

One of the important findings of this study was that the fibres itself
had no effect on the extracellular levels of neurotransmitters or on the
membrane potential of nerve terminals (Table 2A and Fig. 4A). In
contrast, its constituent, DOTAGA, alone induced a concentration-
dependent increase in both glutamate and GABA extracellular levels,
while also depolarizing the plasma membrane of nerve terminals.
Therefore, the chemical grafting of DOTAGA onto chitosan and the as-
sociation of chitosan (via co-crystallization) within the fibre help pre-
vent the potential neurotoxic effects of DOTAGA. These findings are
consistent with our recent data on the action of DOTAGA, both alone
and as a component of gadolinium-based AGuIX nanoparticles used for
the treatment of brain metastases. At high concentrations, the compo-
nents DOTAGA and mixture DOTAGA chelated with Gd(III) significantly
affected synaptic neurotransmission, whereas the AGUIX nanoparticle structure itself mitigated the neurotoxic effects of these constituents [58]. This important experimental observation is critical for building new therapeutic strategies aimed at chelating iron in Parkinson's disease using deferiprone. Oral deferiprone actually worsened the clinical picture of patients in a double-blind controlled study [59], an effect that could be attributed to its own direct activity in neurotransmitters. Although it is challenging to compare precisely the number of free DOTAGA molecules in solution or those grafted onto chitosan in our study, the attachment of DOTAGA to the fibre prevents any toxic effects within cells at local and distant locations while maintaining its efficacy in chelating several metal ions. Our results on the membrane potential of nerve terminals presented in Fig. 4A are consistent with the literature data, where the fibre constituent, chitosan *per se* sealed damaged membranes of nerve cells, restored the conduction of nerve impulses, thereby acting as a neuroprotector in spinal cord trauma and brain injury [60]. Results presented in Table 2A on the extracellular levels of neurotransmitters agreed with the literature data on the protective role of chitosan nanoparticles, in particular improvement in the levels of neurotransmitters, mitochondrial biogenesis, and oxidative stress, shown in rats treated with hydroxyapatite nanoparticles [61]. Dose dependence of DOTAGA effects in nerve terminals within the concentration range of 0.5–5 mM, and increased extracellular neurotransmitter levels and plasma membrane depolarization starting from a concentration of 1.25 mM (Table 2B and Fig. 4B) may be of value for prognosis of potential side effects from impaired synaptic neurotransmission during DOTAGA therapy, in particular, in neoadjuvant radionuclide therapy of patients with glioblastomas that use the peptidic vector 90Y-DOTAGA-substance P [62].

The fibre effectively mitigated the metal-induced increase in the extracellular level of L-[¹⁴C]glutamate and [³H]GABA in nerve terminals, showing the highest efficiency against the effects induced by Cu(II) and Pb(II), followed by Cd(II). The beneficial effect of the fibre was attributed to the chelation properties of the grafted chelator DOTAGA, acknowledging that various metal ions could competitively bind to the chelator. The identity of the metal predominantly bound by a chelating agent depends on factors such as the accessibility of the chelator to the tissues, the strength of the metal's existing binding within the tissues, the metal's affinity for the chelator, and, to some extent, the relative concentrations of various ions [14]. As demonstrated by the ICP-MS analysis of the chelation experiment, the fibre exhibited the ability to chelate Cu (II), Cd(II), and Pb(II) in a physiological solution. Additionally, the mitigation of the increased extracellular release of both excitatory and inhibitory neurotransmitters was dependent on the length of the fibre, with greater effectiveness observed for longer fibre sizes. The efficacy of the fibre may also be attributed to the antioxidant properties of Chitosan@DOTAGA, which were previously shown in another study [23]. However, the fibre was unable to reduce Hg(II)-induced increases in the extracellular levels of both neurotransmitters in nerve terminals. Several studies have reported that mercury elevates glutamate concentration at the synaptic cleft [58] and may enhance oxidative metabolism. Since chelation of Hg(II) by the fibre was not significant, this suggests that the antioxidant properties of the Chitosan@DOTAGA fibre may not be sufficient to counteract the detrimental effects of Hg(II). Therefore, the lack of effect of the fibre against Hg(II)-induced increases in the extracellular levels of glutamate and GABA is likely due to the stronger interaction of Hg(II) with synaptosome membrane components and the weak affinity of DOTAGA for Hg(II) [50,63,64]. Furthermore, given the absence of impact of the fibre on neurotransmitters levels regardless of its length (0.5 or 5 cm), it can be suggested that the fibre did not significantly affect the concentrations of essential physiological metal ions, such as Ca (II) or Mg(II), which are crucial for neurotransmission,

The feasibility of inserting the fibre into the brain of animal models to study their behavior over weeks are fundamental steps towards any potential clinical application. The present results showed that the implantation of chelating fibres in the rat striatum could be well tolerated

up to 17 weeks. Magnetic resonance imaging showed that the chelating fibre can be accurately inserted into the striatum under stereotactic conditions. It was observed that the fibre did not overextend once implanted into the brain. Instead, it was found that its initial area measured the day of implantation was stable during 7 weeks and then progressively shrank to reach 60% of its initial area at the completion of the experiment (17 weeks). The incorporation of gadolinium ions in a fraction of the DOTAGA chelates proved to be useful to improve the MRI contrast between the chelating fibre and the surrounding brain tissue. In line with the thickness reduction of the fibre at completion of the experiment, ICP-MS analysis of the brain hemispheres reveals that 46% of the total Gd was lost within the fibre's zone (right hemisphere). These findings strongly suggested that the fibre was biodegradable, DOTAGA-M complexes being stage and eliminated by the kidneys. Moreover, no acute or subchronic inflammatory reaction due to the presence of the fibre could be observed in any type of MRI image in vivo as well as under subsequent histological examination of the tissue ex vivo. These findings were in agreement with the known good biocompatibility of the chitosan polymer, and its application for the design of neuro-regenerative implants in the central nervous system [20,65,66].

## 5. Conclusion

In summary, the present study demonstrated that grafting DOTAGA onto chitosan results in a polymer that can be mixed with conventional chitosan to obtain a neurocompatible biomaterial. The resulting fibre effectively reduced the damaging effects of Cu(II), Cd(II), and Pb(II) on key parameters of synaptic neurotransmission in nerve terminals, although it had no impact on Hg(II). The protective properties of the fibre can be attributed, at least in part, to its chelating ability, which also can explain its lack of therapeutic effect in the Hg(II) experiments, but it's interesting to see that it's higher than molecular DOTAGA.

A first *in vivo* proof of feasibility demonstrated that the fibre can be safely implanted in the brain of rodent with no signs of neurotoxicity. Additionally, MRI and ICP-MS analyses of an implanted Gd(III)-marked fibre confirmed its biodegradability, with approximately half of the fibre degrading over the implantation period. These findings pave the way for further preclinical studies using appropriate animal models of neurological disorders associated with metal dyshomeostasis, with the goal of assessing the fibre's *in vivo* chelation capacity and its potential associated therapeutic effect. Future studies should also evaluate the behavioural impact of fibre implantation in animal models, as well as the bio-distribution of the degraded fibre.

## CRediT authorship contribution statement

**Tatiana Borisova:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Conceptualization. **Juan Felipe Salazar Ariza:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. **Natalia Pozdnyakova:** Investigation, Formal analysis, Data curation. **Natalia Krisanova:** Investigation, Formal analysis, Conceptualization. **Artem Pastukhov:** Investigation, Formal analysis, Data curation. **Marina Dudarenko:** Visualization, Investigation, Data curation, Conceptualization. **Thomas Gréa:** Validation, Methodology, Investigation. **Alexandra Montembault:** Validation, Supervision, Methodology, Conceptualization. **Imad Benkhaled:** Visualization, Methodology, Investigation. **Noël Pinaud:** Validation, Methodology, Investigation. **Justine Deborne:** Methodology, Investigation, Data curation. **Yannick Crémillieux:** Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. **Philippe De Deurwaerdere:** Writing – review & editing, Validation, Methodology, Investigation, Conceptualization. **Vladimir Lysenko:** Writing – review & editing, Supervision, Methodology, Conceptualization. **Stéphane Palfi:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Conceptualization. **Olivier Tillement:** Writing – review & editing, Validation,
Supervision, Resources, Project administration, Methodology, Conceptualization. **Laurent David:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization. **François Lux:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Conceptualization.

## Authors' contributions

TB, VL, YC, LD, FL, OT – conceptualisation; AP – isolation of synaptosomes; AP, NK, NP – L-[¹⁴C] glutamate measurements and fluorimetry; NP, MD, NK – [³H] GABA measurements; NP, NK, TB, LD, FL – data analysis; JFAS, NP, LD, YC, FL – figure preparation; TB–the design of the neurochemical study; TB, JFAS LD, FL – draft manuscript writing; NP, JD, IB and YC – the design, implementation, execution and analysis of the in vivo and MRI study. The manuscript was edited by TB, JPSA, PD, FL, LD, OT. All authors read and approved the final manuscript.

## Declaration of competing interest

The authors declare no financial and non-financial competing interests exist.

## Acknowledgements

This work was supported by the National Research Foundation of Ukraine, Project #2021.01/0061 (AP, MD, TB - neurochemistry study using Hg, Cd and Pb), Project of Polish Academy of Sciences within the "Long-term program to support Ukrainian research teams" PAN.BFB.S. BWZ.380.022.2023 (NK, TB - neurochemistry study using Cu), and EU Horizon 2020 Research and Innovation Staff Exchange Programme (RISE) under Marie Skłodowska-Curie Action (project 101008159 "UNAT"). The work was supported by French Agence Nationale de la Recherche ANR (ANR SEMB ANR-20-CE19-0011). The authors acknowledge MexBrain company for furnishing chitosan based polymer.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2025.144346.

## Data availability

All study data are included in the article and/or supporting information.

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