Synthesis of carboxymethyl chitosan-coated magnetite nanoparticles and their protective effect against manganese ion-induced damage in human neuroblastoma SH-SY5Y cells

Abstract

Abstract

Background:Excessive intake of manganese can accumulate in the body, causing damage to the nervous system and triggering a series of serious medical problems. Finding effective methods to remove excess manganese ions from the body is crucial for related diseases. It aimed to prepare carboxymethyl chitosan (CMCS)-coated magnetite nanoparticles (Fe₃O₄ NPs) (CMCS-Fe₃O₄ NPs) and investigate their effects on human neuroblastoma SH-SY5Y cells. Methods: Fe₃O₄ NPs were prepared using the co-precipitation method and coated with CMCS to obtain CMCS-Fe₃O₄ NPs. Simulated manganese ion wastewater solutions of different concentrations were prepared for adsorption experiments. SH-SY5Y cells were used to construct a nerve cell damage model, with cells grouped: blank group (BG), model group (MG), and intervention group (IG, treated with CMCS-Fe₃O₄ NPs solution). Multiplication activity, reactive oxygen species (ROS) content, apoptosis rate (AR), and transfer and attack capabilities were recorded. With increasing initial manganese ion concentration, the adsorption capacities of both CMCS-Fe₃O₄NPs and Fe₃O₄ NPs increased, with the former consistently exhibiting higher values (maximum experimental saturated adsorption capacity: 118.3 mg/g). The particle size of CMCS-Fe₃O₄ NPs (53–99 nm) was larger than that of Fe₃O₄ NPs (22–50 nm), but the uniformity of distribution did not improve. The zeta potential became more negative (−30.08 ± 0.08 mV), and superparamagnetism was retained (saturation magnetization: 65.2 emu/g). Compared with the BG group, the MG group showed reduced cell proliferation, increased apoptosis, decreased migration and invasion abilities, and a significant increase in ROS level to 318.52 ± 11.36 (P < 0.01). In contrast, the IG group exhibited increased proliferation, decreased apoptosis, and enhanced migration and invasion capacities compared to the MG group (P < 0.05), along with a reduction in ROS level to 182.47 ± 7.93 (P < 0.01). CMCS-Fe₃O₄ NPs exhibit excellent adsorption capacity for manganese ions and alleviate manganese-induced damage in SH-SY5Y cells through dual mechanisms of adsorbing manganese ions and scavenging ROS, demonstrating potential application value in the prevention and treatment of manganese-related neurotoxic diseases. The innovation of this study lies in the first application of CMCS-Fe₃O₄ NPs in repairing manganese-induced neuronal cell injury. By precisely optimizing the mass ratio of CMCS to Fe₃O₄ NPs and the coating process parameters, the composite material retains the superparamagnetism of Fe₃O₄ NPs while significantly enhancing the adsorption capacity for manganese ions and maintaining excellent adsorption stability within the physiological pH range. This work provides a novel functional material and experimental basis for the targeted treatment of manganese poisoning.

Graphical abstract

Histogram of PS distribution of CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs. (A for CMCS-Fe₃O₄ NPs; B for Fe₃O₄ NPs)

Background

In the field of medical research, with the in-depth exploration of the relationship between environmental factors and human health, the impact of heavy metal pollution on human physiological functions has become a research hotspot. Manganese, as an element with dual properties, holds a unique position in medicine. An appropriate amount of manganese is an essential component of many enzymes in the human body, playing an indispensable role in bone development, the synthesis and metabolism of neurotransmitters, etc. [1, 2]. For example, manganese superoxide dismutase (Mn-SOD) can effectively eliminate superoxide anion radicals in the body, maintaining the redox balance within cells and protecting cells from oxidative damage. However, once the body ingests excessive manganese, this delicate balance is disrupted, leading to serious medical problems. In clinical practice, manganese poisoning cases are mostly concentrated in populations with long-term occupational exposure, such as those involved in manganese mining and welding operations. Excessive manganese accumulates in the body, selectively damaging the nervous system, particularly the basal ganglia and brainstem [3]. Early symptoms manifest as neurasthenia-like symptoms, such as insomnia, emotional fluctuations, and memory impairment, which are closely related to the imbalance of brain neurotransmitters. Metabolic disorders of neurotransmitters such as serotonin and dopamine lead to abnormal nerve conduction, thereby affecting sleep and emotional regulation. As the degree of poisoning deepens, patients develop typical extrapyramidal symptoms, such as raised muscle tone, bradykinesia, and tremors, which are extremely similar to the clinical manifestations of Parkinson’s disease. However, unlike Parkinson’s disease, the neurotoxic mechanisms of manganese poisoning are more complex, involving multiple pathological processes such as oxidative stress damage, mitochondrial dysfunction, and neuronal apoptosis [4, 5]. In subsequent experiments, the intervention effects of carboxymethyl chitosan (CMCS)-Fe₃O₄NPs on these toxic mechanisms will be further verified by detecting intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential, among other indicators. Excessive ROS generated by oxidative stress can attack lipids, proteins, and nucleic acids on neuronal cell membranes, causing structural and functional damage. Mitochondria, as the “energy factories” of cells, can lead to insufficient energy supply in neuronal cells when dysfunctional, affecting normal metabolism and function. Neuronal apoptosis is one of the key factors contributing to irreversible damage in the nervous system.

In the exploration of medical treatments for manganese poisoning, current clinical approaches primarily rely on chemical chelating agents, yet their limitations have motivated the development of novel adsorption materials. Clinically used aminopolycarboxylate chelators such as calcium disodium edetate and calcium trisodium pentetate, though capable of forming stable complexes with manganese ions via carboxyl and amino groups, suffer from poor chelation selectivity. They readily bind essential metal ions such as calcium and zinc, potentially causing electrolyte imbalances and renal damage with long-term use. Moreover, their inability to cross the blood-brain barrier limits their effectiveness in removing manganese accumulated in the central nervous system. Sulfhydryl-based antidotes like sodium dimercaptosuccinate exhibit high efficiency in clearing manganese from peripheral tissues such as the liver and kidneys but are similarly restricted by the blood-brain barrier. Some patients also experience gastrointestinal adverse effects such as nausea and vomiting. Sodium para-aminosalicylate, as an auxiliary detoxifying agent, only promotes the biliary excretion of manganese and demonstrates limited efficacy when used alone. It cannot address manganese accumulation or damage in neural tissues. Furthermore, these traditional metal chelators are mostly small-molecule drugs with rapid metabolism, requiring repeated administration to maintain effective concentrations. They only remove free manganese ions and fail to repair existing neuronal damage, reflecting a limitation of “clearance without repair.” Against this backdrop, the search for efficient and safe adsorption materials has become crucial to overcoming the bottlenecks of conventional therapies. Research on heavy metal ion adsorption has evolved over years, with various adsorption materials exhibiting distinct characteristics in application scenarios and adsorption performance due to differences in structure and properties. In early studies, activated carbon was a representative traditional adsorption material due to its high specific surface area, capable of binding heavy metal ions through physical adsorption. However, its poor selectivity, susceptibility to pH influence, and difficulty in targeted recovery limited its clinical value in manganese poisoning treatment, as it could cause secondary pollution in vivo [6, 7]. Inorganic mineral materials such as montmorillonite and zeolite adsorbed heavy metal ions via their layered structures and ion exchange capacity, offering low cost and environmental friendliness. Yet, their limited adsorption capacity, poor dispersibility, and biocompatibility in biological systems hindered their ability to reach neural tissues where manganese accumulates via blood circulation [8]. Among organic polymer materials, unmodified chitosan possessed certain chelation ability but suffered from poor water solubility and tendency to agglomerate in physiological environments, reducing adsorption efficiency. In contrast, derivatives modified with functional groups such as carboxyl or amino groups (e.g.,CMCS, hydroxypropyl chitosan) demonstrated improved performance. CMCS, a product of natural biopolymer chitin through carboxymethylation modification, not only exhibited significantly enhanced water solubility and biocompatibility but also formed stable chelates with heavy metal ions via functional groups, greatly improving adsorption selectivity and capacity. Thus, CMCS has become a research hotspot in biomedical adsorption materials in recent years [9]. The emergence of nanomaterials has brought new breakthroughs in the field of heavy metal adsorption. Metal oxide nanoparticles (e.g.,Fe₃O₄ NPs, TiO₂ NPs), owing to their small size, large specific surface area, and abundant surface active sites, exhibit significantly higher adsorption efficiency than traditional bulk materials. Among them, Fe₃O₄ NPs, with their unique superparamagnetic properties, enable precise localization and separation via external magnetic fields, reducing damage to normal tissues and demonstrating irreplaceable advantages in targeted in vivo adsorption [10, 11]. However, the stability and biocompatibility of Fe₃O₄ NPs in vivo remain major bottlenecks for their application. Their tendency to aggregate not only reduces adsorption efficiency but may also lead to NPdeposition and accumulation in vivo, potentially causing adverse effects such as inflammation. To address the limitations of single-component materials, composite nano-adsorbents have become a research trend. Combining the biocompatibility of organic polymers with the adsorption performance and magnetic responsiveness of inorganic NPsallows for complementary performance enhancement. For instance, chitosan-coated Fe₃O₄ NPs composites have improved the biocompatibility of Fe₃O₄ NPs to some extent, yet the chelation capacity of chitosan itself remains limited. In contrast, CMCS, as a derivative of chitosan, introduces more carboxyl groups, significantly enhancing its chelation ability. Moreover, CMCS exhibits better water solubility and maintains excellent dispersibility in physiological environments [12, 13].

In-depth research on the preparation process of CMCS-Fe₃O₄ NPs and their effects on human neurobehavioral functions after adsorbing manganese ions is of great significance in the medical field. Theoretically, this helps to reveal the molecular mechanisms of manganese neurotoxicity, providing new perspectives and research methods for neuroscience. By investigating the interactions between manganese ions and the composite material during the adsorption process and the effects of these interactions on neuronal signaling pathways and gene expression profiles, a deeper understanding of the pathogenesis of manganese poisoning can be achieved. From a practical application standpoint, developing an effective adsorbent material for manganese poisoning can improve quality of life for patients and prevent the occurrence of occupational manganese poisoning. This article focused on optimizing the preparation process of the material, testing its adsorption performance in vitro and in vivo, and assessing its effects on neurobehavioral functions, aiming to provide innovative strategies and means for the medical prevention and treatment of manganese poisoning. Current research has predominantly focused on the individual applications of either Fe₃O₄ NPs or CMCS, leaving significant gaps in the study of composite nanomaterials for manganese poisoning treatment. The innovative aspects of this study are primarily reflected in the following three areas: First, to address the issues of aggregation and poor biocompatibility of traditional Fe₃O₄ NPs, this study designed and optimized a preparation system for CMCS-Fe₃O₄ NPs. By precisely controlling key coating parameters, we constructed composite nanomaterials with a stable core-shell structure, enhancing their dispersibility and safety in biological environments and laying the foundation for subsequent in vivo applications. Second, moving beyond the current research limit of adsorption performance testing under single conditions, this study systematically investigated the influence of various environmental factors (e.g., manganese ion concentration, pH) on the adsorption behavior of CMCS-Fe₃O₄ NPs and elucidated the underlying adsorption mechanisms. This provides theoretical support for the material’s functionality in complex in vivo environments. Third, this study innovatively applied CMCS-Fe₃O₄ NPs to a manganese-induced neuronal injury model. By validating their protective effects on neuronal cells from the perspective of functional recovery, this study addresses the research gap in the application of such composite nanomaterials for neural repair in manganese poisoning, offering critical experimental evidence for future animal studies and clinical translation.

Materials and methods

Chemical reagents and instruments

Ferric chloride hexahydrate (FeCl₃·6H₂O, analytical grade, Beijing Solarbio Science & Technology Co., Ltd.), ferrous chloride tetrahydrate (FeCl₂·4H₂O, analytical grade, Beijing Solarbio Science & Technology Co., Ltd.), ammonia solution (25–28%, chemical pure, Beijing Solarbio Science & Technology Co., Ltd.), CMCS(molecular weight 50,000–100,000 Da, substitution degree ≥80%, biochemical grade, Shanghai Lingjiu Medical Technology Co., Ltd.), manganese sulfate (MnSO₄, analytical grade, Shanghai Lingjiu Medical Technology Co., Ltd.), acetic acid (10%, analytical grade, Shanghai Lingjiu Medical Technology Co., Ltd.), hydrochloric acid (HCl, 36–38%, analytical grade, Beijing Solarbio Science & Technology Co., Ltd.), sodium hydroxide (NaOH, analytical grade, Beijing Solarbio Science & Technology Co., Ltd.), sodium chloride (NaCl, analytical grade, Beijing SolarbioScience & Technology Co., Ltd.), oleic acid (analytical grade, Beijing Solarbio Science & Technology Co., Ltd.), polyethylene glycol (PEG 4000, analytical grade, Beijing Solarbio Science & Technology Co., Ltd.), 0.22 μm polyvinylidene fluoride (PVDF, MembranTech Biotechnology Co., Ltd.) filter membrane, 0.25% trypsin-EDTA solution (Shanghai Yuanpei Biotechnology Co., Ltd.), penicillin-streptomycin solution (100×, penicillin 10,000 U/mL, streptomycin 10,000 μg/mL, Shanghai Yuanpei Biotechnology Co., Ltd.), fetal bovine serum (FBS, qualified grade, Shanghai Yuanpei Biotechnology Co., Ltd.), high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Shanghai Yuanpei Biotechnology Co., Ltd.), CCK-8 assay kit (Shanghai Yuanpei Biotechnology Co., Ltd.), and Annexin V-FITC/PI apoptosis detection kit (Shanghai Yuanpei Biotechnology Co., Ltd.) were used.

Electronic analytical balance (accuracy 0.0001 g, Shanghai Jinghai Instrument Co., Ltd.), magnetic stirrer (Shanghai Jinghai Instrument Co., Ltd.), high-speed centrifuge (Shanghai Jinghai Instrument Co., Ltd.), vacuum drying oven (Shanghai Jinghai Instrument Co., Ltd.), dynamic light scattering (DLS) analyzer (Suzhou Boke Analysis Technology Service Co., Ltd.), JEOL JEM-2100 transmission electron microscope (TEM, Suzhou Boke Analysis Technology Service Co., Ltd.), Lake Shore 7410 vibrating sample magnetometer (Lake Shore Cryotronics, Inc., USA), atomic absorption spectrophotometer (Shanghai Precision Instrument Co., Ltd.), inductively coupled plasma optical emission spectrometer (Shanghai Precision Instrument Co., Ltd.), and CO₂ incubator (Shanghai Beyin Biotech Co., Ltd.), flow cytometer (Shanghai Precision Instrument Co., Ltd.)were used.

Preparation of Fe₃O₄NPs

Fe₃O₄ NPs were prepared using the co-precipitation method. First, appropriate amounts of chemically pure FeCl₃·6H₂O and FeCl₂ ·4H₂O were precisely weighed and placed into a clean three-necked flask at a molar ratio of 2:1. An adequate amount of deionized water was added, and magnetic stirring was initiated at a rate of 600 rpm for 20 min until the solutes were fully dissolved. Subsequently, under nitrogen protection, 2–3 mol/L ammonia solution was slowly added at 1–2 mL/min while continuously stirring and monitoring the color and temperature changes of the solution. The solution gradually changed from light yellow to black, indicating the formation of NPs. After the ammonia solution was completely added, the temperature was raised to 70–80 °C, and stirring continued for an additional 30–40 min to ensure a more complete precipitation reaction. At the end of the reaction, the NPs were separated using an external magnetic field. The supernatant, which contained any unbound or excess materials, was carefully removed to ensure that only the NPs remained. Subsequently, the NPs were thoroughly washed with deionized water at least three times to eliminate any residual impurities or unreacted substances that might interfere with subsequent experiments or applications. This meticulous washing procedure was essential for obtaining pure and well-isolated NPs. Each time, the solid-liquid separation was achieved using the external magnetic field. Finally, the NPs were dispersed in dispersants such as oleic acid and polyethylene glycol (at 10 −20% of the NPs mass) and ultrasonicated for 15–20 min to obtain a stable dispersion, which was prepared for the subsequent coating with CMCS. The prepared NPs were denoted as Fe₃O₄ NPs.

Preparation of CMCS-Fe₃O₄NPs

After preparing a stable dispersion of Fe₃O₄ NPs, the coating with CMCS was initiated. First, an appropriate amount of CMCS powder was precisely weighed based on the mass of the NPs at a mass ratio of 1:2 −1:3. The powder was dissolved in an appropriate amount of acetic acid solution (concentration controlled at 1% −2%) and stirred magnetically for about 30 min at a speed of 500 rpm to form a uniform CMCS solution. Subsequently, the prepared Fe₃O₄ NPs dispersion was slowly added dropwise into the CMCS solution under constant temperature stirring conditions (temperature maintained at 40–50 °C, dropwise speed controlled at 0.5–1 mL/min) to ensure a uniform and stable coating process. After the addition was complete, the mixture was stirred at this temperature for an additional 2–3 h to allow the CMCS to fully coat the surface of the Fe₃O₄ NPs.

After the reaction was completed, the mixed solution was centrifuged using a high-speed centrifuge at a speed of 10,000 rpm for 15 min to collect the precipitate. The precipitate was then washed multiple times with deionized water to remove unreacted CMCS and other impurities (at least three times). The washed precipitate was ultrasonicated for 10–15 min to obtain a stable dispersion of CMCS-Fe₃O₄ NPs, which was used for subsequent experiments such as adsorption of manganese ions. The prepared CMCS-Fe₃O₄ NPs were denoted as CMCS-Fe₃O₄ NPs.

The NP size distribution and Zeta potential were determined using dynamic light scattering (DLS, Malvern Zetasizer Nano ZS) under the following conditions: laser wavelength of 633 nm, scattering angle of 173°, temperature of 25 °C, and sample concentration of 0.1 mg/mL. Each sample was measured in triplicate, and the average value was taken. The morphology of the NPs was observed via transmission electron microscopy (TEM, JEOL JEM-2100). A drop of the sample was placed on a carbon-coated copper grid, allowed to dry naturally, and imaged at an accelerating voltage of 200 kV. Particle size distribution was analyzed by randomly selecting five fields of view.

Magnetic properties were measured using a vibrating sample magnetometer (VSM, Lake Shore 7410). After drying, the samples were compressed into pellets and subjected to hysteresis loop scanning at room temperature. The magnetic field ranged from −10 kOe to 10 kOe with a scan rate of 1 kOe/s. Each sample was measured in triplicate, and the average values for saturation magnetization, coercivity, and remanent magnetization were calculated.

Preparation of simulated manganese ion wastewater solution

Analytical-grade manganese sulfate (MnSO₄) was selected as the source of manganese ions. Based on the desired concentration range of manganese ions in the simulated wastewater, an appropriate amount of MnSO₄ powder was precisely weighed and placed into a clean volumetric flask. For example, to prepare a simulated manganese ion wastewater solution with a concentration of 10 mg/L–100 mg/L, the required mass of MnSO₄ was calculated according to the stoichiometric relationship. An adequate amount of deionized water was then added to the volumetric flask, and the solution was stirred using a magnetic stirrer at a rate of 200–300 rpm for approximately 20 min to ensure complete dissolution of MnSO₄ and uniformity of the solution. To simulate the complex components that may be present in real wastewater, common inorganic anions such as Cl⁻ and SO₄²⁻ could be selectively added to the solution. The addition concentrations were determined based on actual wastewater survey data, with Cl⁻ concentrations typically set at 10 mmol/L −50 mmol/L and SO₄²⁻ concentrations at 20 mmol/L–80 mmol/L. The solution was then diluted to the mark with deionized water, mixed thoroughly, and the pH was measured using a pH meter. The pH was adjusted to the neutral range of 6–8 using dilute hydrochloric acid or sodium hydroxide solution. Finally, the prepared simulated manganese ion wastewater solution was transferred to a brown reagent bottle and stored in a cool, dry place, protected from light and significant temperature fluctuations, for subsequent adsorption experiments.

Adsorption experiment

A certain amount of CMCS-coated magnetite NPs dispersion was added to a series of 100 mL conical flasks containing simulated manganese ion wastewater solutions at varying initial concentrations (10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L). The conical flasks were placed in a thermostatic shaker set to a temperature of 25 °C and an agitation rate of 180 rpm for 2 h. During the adsorption process, 3 mL of the solution was taken every 30 min using a pipette, rapidly filtered through a 0.22 μm PVDF membrane, which had been pre-soaked in 5% nitric acid for 24 h and then rinsed with ultrapure water to a pH of 7. For the 50 mg/L concentration group, a parallel treatment was performed by centrifuging the sample at 10,000 rpm for 10 min. The filtrate or supernatant was collected and the manganese ion concentration was measured using atomic absorption spectroscopy (AAS). The changes in manganese ion concentration during adsorption were monitored in real time, and the adsorption kinetics curve was plotted. After adsorption, the remaining solution was again processed using the filtration method described above, and the final manganese ion concentration in the filtrate was determined. The adsorption capacity of the NPs for manganese ions was calculated using the equation $q=\frac{(C_0-C_t)*V}{m}$. Additionally, several control experiments were conducted, where the pH of the solution was adjusted to 4, 5, 6, 7, 8, and 9 using 0.1 mol/L HCl or NaOH solutions, and the adsorption experiment was repeated as described.

In addition to different concentrations, multiple control experiments were set up by changing the solution pH values [4–9] and repeating the above adsorption experiment steps.

Neural cell injury model

Human neuroblastoma SH-SY5Y cells (purchased from Nanjing Shenghang Biotechnology Co., Ltd.) were selected. The cryopreserved SH-SY5Y cells, which had been stored in liquid nitrogen to maintain their viability, were carefully retrieved and promptly transferred to a 37 °C water bath. This step was crucial for rapid thawing of the cell suspension, ensuring that the cells were revived quickly and uniformly. As the temperature of the water bath facilitated the thawing process, the cells gradually returned to a state suitable for further handling and culturing. Upon near-complete thawing of the cell suspension, it was transferred to a centrifuge tube, followed by the addition of an appropriate volume of pre-warmed complete culture medium (comprising 10% serum and 1% penicillin-streptomycin solution). To ensure a uniform and viable cell suspension, the cells were subjected to centrifugation at 1200 rpm for 5 min. This process effectively concentrated the cells by separating them from the surrounding medium. Following centrifugation, the supernatant, which contained any unbound components or debris, was carefully removed to avoid reintroducing impurities into the cell suspension. Subsequently, the cell pellet was gently resuspended in fresh complete culture medium using a pipette. This step was performed with care to ensure that the cells were evenly distributed within the medium, thereby facilitating their subsequent growth and maintenance in culture. The cells were then seeded into culture flasks and incubated at 37 °C, 5% CO₂ to allow recovery and growth. When the cells achieved 80–90% confluence, indicating that they had reached an optimal density for further propagation, the passaging procedure was initiated. The old culture medium was carefully removed to prepare the next steps, and the cells were subjected to three rinses with sterile phosphate-buffered saline (PBS) to remove residual medium and dead cells. This thorough rinsing process was essential to remove any residual culture medium and dead cells that might interfere with subsequent treatments or the cells’ normal growth. Each rinse with PBS ensured a clean environment for the cells, setting the stage for the next phase of their cultivation. Subsequently, an appropriate amount of 0.25% trypsin-EDTA solution was added, and the cells were digested for 2 min at 37 °C, 5% CO₂. When the cells became round and began to detach, an appropriate amount of complete culture medium was immediately added to terminate the digestion. The cells were gently pipetted to form a single-cell suspension, which was then seeded into new culture flasks at a 1:5 ratio and continued to be cultured.

A 50 mg/L manganese ion solution was used to treat SH-SY5Y to construct a neural cell injury model. The cells were seeded into 6-well plates at a density of 1 × 10⁵ per well. When the cells achieved 8–90% confluence, signaling that they had reached a suitable density for further experimental manipulation, the original culture medium was carefully removed to eliminate any factors that might influence the subsequent treatment. Following this, the cells were exposed to a culture medium specifically formulated to contain the corresponding concentration of manganese ions. This treatment was maintained for a period of 24 h, during which the cells were incubated under standard culture conditions to allow the manganese ions to exert their effects on the cellular environment. This step was crucial for establishing the experimental conditions necessary to investigate the impact of manganese ions on the cells’ behavior and function.

Cell grouping

Cells were grouped: the blank group (BG, normal cells), the model group (MG, neural cell injury model), and the intervention group (IG, treated with CMCS-Fe₃O₄ NPs solution). After 24 h of treatment in the MG, the CMCS-Fe₃O₄NPs solution was diluted to an appropriate concentration with serum-free culture medium. To ensure the desired concentration of NPs was achieved in the culture environment, an appropriate volume of the diluted NP solution was carefully added to the wells of the MG. This step was critical for maintaining a consistent and effective exposure of the cells to the NPs throughout the experiment. Subsequently, the cells were placed in a 37 °C incubator with an atmosphere of 5% CO₂, which are standard conditions for maintaining optimal cell viability and growth. To ensure the cells remained in a healthy state and to maintain the stability of the experimental conditions, the culture medium was systematically replaced every 24 h. This regular medium change helped to remove any metabolic waste products accumulated by the cells and replenished the essential nutrients required for continued cell growth and NPs interaction. After the predetermined duration of 48 h, the recovery of cell morphology and viability was recorded. Multiplication activity was assessed using the CCK-8 assay, AR was measured by flow cytometry, and cell transfer and attack capabilities were evaluated using scratch and Transwell chamber assays, respectively.

Regeneration performance test of CMCS-Fe₃O₄ NPs

0.1 g of Mn²⁺-saturated CMCS-Fe₃O₄ NPs was added to 50 mL of 0.1 mol/L HCl solution and oscillated at 25 °C and 150 r/min for 2 h for desorption. The material was magnetically separated, washed with deionized water to neutrality, and vacuum-dried at 60 °C. The “adsorption (50 mg/L Mn²⁺solution, pH=7.0, 25 °C, 2 h) - desorption” cycle was repeated 5 times. The Mn²⁺ concentration after each adsorption was measured by atomic absorption spectrophotometry to calculate the adsorption capacity and desorption rate.

Long-term stability test of CMCS-Fe₃O₄ NPs

0.1 g of CMCS-Fe₃O₄ NPs was placed in three 50 mL systems (deionized water; simulated wastewater: 50 mg/L Mn²⁺ + 0.1 mol/L NaCl, pH=7.0; physiological saline) and stored statically at 25 °C for 30 days. Samples were taken every 5 days to measure particle size distribution and zeta potential by dynamic light scattering (DLS), and Fe ion leaching was detected by inductively coupled plasma optical emission spectrometry (ICP-OES). The material was observed for aggregation, precipitation, and visual changes.

Intracellular ROS level detection

SH-SY5Y cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 5×10³ cells per well, with 100 μL of high-glucose DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) added to each well. The cells were cultured at 37 °C in a 5% CO₂ incubator for 24 h. According to the grouping, treatments were applied as follows: BG: medium only; Manganese ion MG: 500 μmol/L MnSO₄; IG: pretreated with 50 μg/mL CMCS-Fe₃O₄ NPs for 2 h, then exposed to 500 μmol/L MnSO₄; Fe₃O₄ NPs alone group: 50 μg/mL Fe₃O₄ NPs. After 24 h of further incubation, 10 μmol/L DCFH-DA fluorescent probe was added to each well and incubated at 37 °C in the dark for 30 min. Cells were gently washed three times with PBS buffer to remove uninternalized probe. Fluorescence intensity was measured using a microplate reader at excitation/emission wavelengths of 488/525 nm. Each group included three replicate wells, and the experiment was repeated three times. The relative fluorescence intensity of ROS was calculated with the BG set as 100%.

Statistical methods

SPSS 22.0 was employed. Measurement data that followed a normal distribution were represented as mean ± SD ($\overline{\mathrm{x}}\pm \mathrm{s}$), and categorical data were represented as frequencies and percentages (%). For measurement data that did not follow a normal distribution, the Mann–Whitney test was adopted. For normally distributed measurement data, one-way analysis of variance was applied, and for categorical data, χ² test was adopted. A two-sided test with P < 0.05 was considered statistically meaningful.

Results

Characterization results of CMCS-Fe₃O₄NPs and Fe₃O₄NPs

Figure 1 presents the particle size distribution histograms of Fe₃O₄ NPs and CMCS-Fe₃O₄ NPs. The logarithmic particle size range (ln) of Fe₃O₄ NPs was 3.091–3.912, corresponding to a size range of 22–50 nm. The logarithmic mean was 3.589 ± 0.213, and after exponential transformation, the geometric mean (GMean) was 36.27 nm, with a geometric standard deviation (GSD) of 1.24 (corresponding to a logarithmic standard deviation of 0.213). The distribution of logarithmic values showed that data were primarily concentrated in the range of 3.466–3.714 (corresponding to particle sizes of 32–41 nm), accounting for 52.2% (24/46) of the total data points, exhibiting a unimodal symmetric distribution with good uniformity. The goodness-of-fit for the log-normal distribution was R² = 0.96, confirming that the particle size distribution followed a log-normal model. For CMCS-Fe₃O₄NPs, the logarithmic particle size range was 3.970–4.595, corresponding to a size range of 53–99 nm, indicating a significant increase in particle size compared to Fe₃O₄ NPs. The logarithmic mean was 4.318 ± 0.235, with a geometric mean of 75.23 nm and a geometric standard deviation of 1.26 (logarithmic standard deviation of 0.235). The highest frequency distribution interval was 4.331–4.443 (corresponding to particle sizes of 76–85 nm), accounting for only 35.7% (15/42) of the total data points, exhibiting a right-skewed distribution, indicating that the uniformity of the particle size distribution of CMCS-Fe₃O₄NPs did not improve compared to Fe₃O₄ NPs. Despite differences in distribution uniformity, the goodness-of-fit for the log-normal distribution remained high (R² = 0.94), validating the applicability of this distribution model.

Fig. 1.

Histogram of PS distribution of CMCS-Fe₃O₄NPs and Fe₃O₄NPs. (A for CMCS-Fe₃O₄NPs; B for Fe₃O₄NPs)

Figure 2 illustrates the Zeta potentials of CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs. The Zeta potential of CMCS-Fe₃O₄ NPs was −30.08 ± 0.08 mV, while that of Fe₃O₄ NPs was −22.56 ± 3.06 mV.

Fig. 2.

Zeta potential of CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs

Figure 3 illustrates the dispersion index. The dispersion index of CMCS-Fe₃O₄ NPs was −30.08 ± 0.08 mV, while that of Fe₃O₄ NPs was −22.56 ± 3.06 mV.

Fig. 3.

Dispersion index of CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs

Figure 4 shows the TEM images of the NPs. The results reveal that Fe₃O₄ NPs are spherical in shape, with particle sizes concentrated in the range of 20–50 nm. In contrast, CMCS-Fe₃O₄ NPs exhibit a distinct core-shell structure, with particle sizes expanded to 50–100 nm, confirming the successful coating of CMCS.

Fig. 4.

TEM images of CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs. (A corresponds to Fe₃O₄ NPs and B corresponds to CMCS-Fe₃O₄ NPs)

Furthermore, magnetic characterization of Fe₃O₄ NPs and CMCS-Fe₃O₄ NPs was performed using a vibrating sample magnetometer (VSM, Lake Shore 7410) under the following test conditions: room temperature (25 °C) and a magnetic field strength range from −10 kOe to 10 kOe. The results show that Fe₃O₄ NPs exhibit a saturation magnetization (Ms) of 78.5 emu/g, a coercivity (Hc) of <10 Oe, and a remanent magnetization (Mr) close to 0, confirming their superparamagnetic characteristics. After CMCS coating, the Ms decreased to 65.2 emu/g, which can be attributed to the dilution effect of the non-magnetic CMCS layer. However, the Hc and Mr remained at very low levels, indicating that the coating did not destroy the superparamagnetic properties.

A critical analysis of the characterization data reveals that while Fe₃O₄NPs exhibit superparamagnetism (saturation magnetization 78.5 emu/g), their particle size range (22–50 nm) and zeta potential (−22.56 mV) predispose them to aggregation. After CMCS coating, the composite demonstrates an average particle size of 75.23 nm, a more negative zeta potential (−30.08 mV), and a distinct core-shell structure. These modifications effectively mitigate aggregation issues while retaining superparamagnetic properties (saturation magnetization 65.2 emu/g). This confirms that CMCS modification simultaneously enhances both the stability and biocompatibility of the material. In summary, the characterized parameters of CMCS-Fe₃O₄NPs meet the requirements for in vivo applications in terms of dispersibility, magnetic responsiveness, and structural stability, thereby providing a solid material foundation for subsequent adsorption and cell experiments. This conclusion is supported by Fig. 4 (TEM images) and VSM results: Fig. 4B clearly illustrates the core-shell structure of CMCS-Fe₃O₄NPs with a uniform shell thickness, confirming successful CMCS coating without compromising the structural integrity of the Fe₃O₄ core.

Adsorption experiment results of CMCS-Fe₃O₄NPs and Fe₃O₄NPs

In Fig. 5, with the increase in the initial concentration of manganese ions, the adsorption capacity of both CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs exhibited a gradual increase. Notably, the adsorption capacity of CMCS-Fe₃O₄ NPs was consistently higher than that of Fe₃O₄ NPs, with the difference being statistically significant (P < 0.05). Specifically, at 10 mg/L, P = 0.002, and at 50 mg/L, P < 0.001.

Fig. 5.

Contrast of AC of CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs at different manganese ion concentrations. Note: * as against CMCS-Fe₃O₄ NPs, P < 0.05

In Fig. 6, as the pH value increased from 4 to 7, the adsorption capacity of both CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs gradually increased. After reaching a maximum, the adsorption capacity began to decrease as the pH continued to rise. Notably, the adsorption capacity of CMCS-Fe₃O₄ NPs was consistently higher than that of Fe₃O₄ NPs, with the difference being statistically significant (P < 0.05). At pH = 7, P = 0.003.

Fig. 6.

Contrast of AC of CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs at different manganese ion concentrations. Note: * as against CMCS-Fe₃O₄ NPs, P < 0.05

Critical analysis of adsorption data under varying concentrations and pH conditions reveals that the increase in adsorption capacity of Fe₃O₄ NPs with rising concentration gradually plateaus due to insufficient surface active sites. In contrast, CMCS-Fe₃O₄NPs, leveraging the synergistic effect of CMCS chelating groups and the high specific surface area of Fe₃O₄, maintain superior adsorption capacity across the entire concentration range (10–50 mg/L) and within the physiological pH range [6–8]. Moreover, their adsorption stability is significantly enhanced compared to Fe₃O₄ NPs, confirming that the optimization of adsorption performance through CMCS modification is environmentally adaptive. In summary, the adsorption advantages of CMCS-Fe₃O₄NPs under simulated physiological conditions demonstrate their potential to eliminate excess manganese ions in vivo, providing functional feasibility for subsequent neuroprotection experiments. This finding is corroborated by Fig. 6 (comparative adsorption capacity under different pH conditions): the adsorption capacity curve of CMCS-Fe₃O₄NPs peaks within the pH 6–8 range and is significantly higher than that of Fe₃O₄ NPs. The largest difference in adsorption capacity between the two occurs at pH = 7 (P = 0.003), aligning perfectly with the conclusion of physiological pH adaptability and robustly supporting the feasibility of applying this material in vivo.

Contrast of multiplication activity and AR

In Fig. 7, compared to the BG, the cell proliferation activity in the MG was significantly reduced, while the AR was increased, with statistically significant differences (P < 0.05). Specifically, the proliferation activity was P = 0.001, and the AR was P < 0.001. In contrast, compared to the MG, the IG exhibited a significant increase in cell proliferation activity and a decrease in AR, with statistically significant differences (P < 0.05). Specifically, the proliferation activity was P = 0.002, and the AR was P = 0.003.

Fig. 7.

Contrast of multiplication activity and AR. (A for multiplication activity; B for AR). Note: * as against the BG, # as against the MG, P < 0.05

Contrast of cell transfer and attack

In Fig. 8, compared to the BG, the cell migration and invasion abilities in the MG were significantly reduced, with statistically significant differences (P < 0.05). Specifically, the migration ability was P = 0.005, and the invasion ability was P = 0.004. In contrast, compared to the MG, the IG exhibited a significant increase in both cell migration and invasion abilities, with statistically significant differences (P < 0.05). Specifically, the migration ability was P = 0.006, and the invasion ability was P = 0.007.

Fig. 8.

Contrast of cell transfer and attack capabilities. (A for transfer; B for attack). Note: * as against the BG, # as against the MG, P < 0.05

Critical analysis of cell proliferation, apoptosis, migration, and invasion data reveals that the toxic effects of manganese ions on SH-SY5Y cells are not limited to mere inhibition of cell activity but involve disruption of metabolic balance (e.g., proliferation-apoptosis imbalance, reduced migration capacity), leading to neural functional impairment. In contrast, the comprehensive restoration of cell function following CMCS-Fe₃O₄NPs intervention is not solely attributable to manganese ion adsorption but is also associated with the synergistic effects of the material in improving the cellular microenvironment and mitigating toxic damage, thereby refuting the simplistic hypothesis of mere physical adsorption. In summary, CMCS-Fe₃O₄NPs can reverse manganese-induced neuronal damage through dual mechanisms, adsorbing manganese ions and protecting cellular function, confirming their practical utility in neuroprotection against manganese poisoning. This conclusion is supported by Fig. 8 (comparative migration and invasion assays): Fig. 8A (scratch assay results) shows a higher wound healing area percentage in the IGcompared to the MG, while Fig. 8B (Transwell assay results) demonstrates a significantly higher percentage of transmigrated cells in the IG(P = 0.007). The graphical data directly validate the restoration of cell migration and invasion capabilities.

Regeneration performance and long-term stability of CMCS-Fe₃O₄ NPs

In Table 1 below, after five adsorption-desorption cycles, the changes in adsorption capacity and desorption rate of CMCS-Fe₃O₄NPs are summarized. The material retained 82.3% of its initial adsorption activity, and the desorption rate remained above 78% without a significant decline.

Table 1.

Regeneration performance test results of CMCS-Fe₃O₄NPs

Numberof cycles	Adsorption capacity (mg/g)	Desorption rate (%)	Adsorption capacity retention rate (%)
1	28.6 ± 0.5	84.7 ± 1.2	100.0
2	26.3 ± 0.4	82.5 ± 0.9	92.0
3	24.5 ± 0.3	80.1 ± 1.0	85.7
4	23.1 ± 0.4	79.3 ± 0.8	80.8
5	23.6 ± 0.3	78.2 ± 0.7	82.3

In Table 2 below, during the 30-day static incubation experiment, the stability parameters of the material in the three systems are summarized. The coefficient of variation for particle size distribution was <5%, the zeta potential remained stable within the range of −28 to −32 mV, and the Fe ion leaching was consistently below 0.05 mg/L. No aggregation or precipitation was observed.

Table 2.

Long-term stability test results of CMCS-Fe₃O₄NPs

System	Testing time (days)	Average particle size (nm)	Particle size variation coefficient (%)	Zeta potential (mV)	Fe ion leaching amount (mg/L)
Deionizedwater	0	75.2 ± 2.1	3.2	−30.1 ± 0.5	Not detected
Deionizedwater	30	77.5 ± 2.3	4.1	−28.6 ± 0.6	0.032 ± 0.005
Simulatedwastewater	0	76.1 ± 1.8	3.5	−29.8 ± 0.4	Not detected
Simulatedwastewater	30	78.3 ± 2.0	4.5	−28.2 ± 0.7	0.041 ± 0.006
Normalsaline	0	75.8 ± 2.2	3.4	−30.3 ± 0.5	Not detected
Normalsaline	30	79.1 ± 2.4	4.8	−29.0 ± 0.6	0.038 ± 0.004

Critical analysis of the regeneration performance and long-term stability data indicates that after five adsorption-desorption cycles, CMCS-Fe₃O₄NPs retained over 80% of their adsorption capacity, with a desorption rate consistently above 78%. This demonstrates that the material can be regenerated through simple acid desorption, addressing the limitation of high single-use costs associated with traditional adsorbents. In the 30-day static incubation experiment, the particle size and zeta potential of the material showed no significant fluctuations across the three systems, and Fe ion leaching was far below safety standards. This confirms its long-term structural stability in both aqueous and physiological environments, effectively avoiding secondary pollution caused by material degradation or ion leaching during use. In summary, the high regenerability and long-term stability of CMCS-Fe₃O₄NPs not only meet the reuse requirements for ex vivo wastewater treatment but also provide safety assurance for potential in vivo applications, further validating the material’s practical performance. This conclusion is supported by Table 1 (regeneration performance data) and Table 2 (long-term stability data): Table 1 clearly illustrates the trends in adsorption capacity and desorption rate across cycles, confirming regenerative potential; Table 2 visually demonstrates the stability of the material in different environments through time-point data on particle size, zeta potential, and leaching, ensuring all conclusions are empirically grounded.

Regeneration performance and long-term stability of CMCS-Fe₃O₄ NPs

As shown in the intracellular ROS level detection results (Table 3), the relative fluorescence intensity of ROS in the BG (high-glucose DMEM medium only, containing 10% fetal bovine serum and 1% penicillin-streptomycin) was 100.00 ± 4.89. In the manganese ion MG(treated with 500 μmol/L MnSO₄ for 24 h), the relative fluorescence intensity of ROS significantly increased to 318.52 ± 11.36, showing a statistically significant difference compared to the BG (P < 0.01). This indicates that manganese ions significantly induced oxidative stress in SH-SY5Y cells, leading to substantial ROS generation. In the CMCS-Fe₃O₄NPs IG(pretreated with 50 μg/mL CMCS-Fe₃O₄NPs for 2 h, then treated with 500 μmol/L MnSO₄ for 24 h), the relative fluorescence intensity of ROS decreased to 182.47 ± 7.93. Although this value remained higher than that of the BG (P < 0.05), it was significantly lower than that of the manganese ion MG (P < 0.01).

Table 3.

Long term stability test results of CMCS-Fe₃O₄NPs

Group	ROS relative fluorescence intensity ($\overline{\mathrm{x}}$ ± s)	Compared with the control group (P value)	Comparison with the MG (P value)
BG	100.00 ± 4.89	–	–
MG	318.52 ± 11.36	<0.01	–
IG	182.47 ± 7.93	<0.05	<0.01

Discussion

In the medical field, the impact of heavy metals on human health has always been a significant and highly concerned issue. Manganese, as one of the essential trace elements in the human body, plays a key role in many physiological processes, such as participating in enzyme activation, maintaining bone development, and nerve conduction [14, 15]. However, when the manganese content in the body exceeds the normal range, excess manganese accumulates in tissues and organs such as the nervous system, triggering a series of serious medical problems, including neurobehavioral dysfunction and Parkinson’s syndrome-like symptoms, which severely affect patients’ quality of life and physical health [16]. Therefore, exploring effective methods to remove excess manganese ions from the body and mitigate their damage to nerve cells has become an important direction in medical research. This article focused on the preparation of CMCS-Fe₃O₄ NPs and their adsorption performance for manganese ions, as well as the effects of this adsorption on human nerve cells. This study employed log-normal distribution fitting for the NP size, based on the following scientific background: during the preparation process, NP growth typically follows an exponential law (such as Ostwald ripening or aggregation processes), and its logarithmic transformation adheres to a normal distribution characteristic. The logarithmic mean of Fe₃O₄ NPs was 3.589 (corresponding to a geometric mean of 36.27 nm), and for CMCS-Fe₃O₄ NPs, it was 4.318 (corresponding to 75.23 nm). Both distributions had a goodness of fit (R²) greater than 0.94, confirming the applicability of the model. This result is consistent with the reported size distribution behavior of chitosan-coated magnetic NPs in the literature, suggesting that CMCS coating did not alter the statistical properties of the size distribution but significantly increased the average particle size [17]. In terms of Zeta potential, the Zeta potential of CMCS-Fe₃O₄ NPs was −30.08 ± 0.08 mV, while that of Fe₃O₄ NPs was −22.56 ± 3.06 mV. Zeta potential is an important indicator for measuring the surface charge properties and stability of NPs. In the body, the stability of NPs is crucial for their functionality. In terms of zeta potential, CMCS-Fe₃O₄ NPs exhibited a zeta potential of −30.08 ± 0.08 mV, while Fe₃O₄ NPs showed −22.56 ± 3.06 mV. Zeta potential is a critical indicator of the surface charge properties and stability of NPs. In vivo, the stability of NPs is essential for their functional performance. The more negative zeta potential of CMCS-Fe₃O₄ NPs primarily stems from the abundant carboxyl (-COOH) functional groups in CMCS molecules. Under physiological conditions (pH 6–8), these carboxyl groups dissociate (-COOH → -COO⁻ + H⁺), imparting a stronger negative surface charge to the NPs. In contrast, amino (-NH₂) functional groups only protonate (-NH₂ + H⁺ → -NH₃⁺) and carry positive charges in low-pH environments, contributing negligibly to negative charge generation within the physiological pH range [18]. This higher surface charge density enhances the stability of the NPsin vivo, reduces aggregation, and facilitates their transport and action toward target cells. It is noteworthy that, although the overall particle size of CMCS-Fe₃O₄ NPs in this study (53–99 nm) exceeds the critical size for superparamagnetism, the core Fe₃O₄ particle size (20–50 nm) still meets the conditions for superparamagnetic behavior. VSM results show that its saturation magnetization (Ms) is 65.2 emu/g, a 17% decrease compared to Fe₃O₄ NPs (78.5 emu/g), which is consistent with the reduced volume fraction of the magnetic core due to the coating layer thickness (estimated to be approximately 15–20 nm by TEM). The retention of superparamagnetism provides experimental support for the directional enrichment of NPs under an applied magnetic field. Combined with their high adsorption capacity, CMCS-Fe₃O₄ NPs exhibit potential application value in magnetic separation for the removal of manganese from contaminated water. Previous studies showed that when the particle size of chitosan-coated magnetic NPs exceeds 50 nm, their magnetic responsiveness decreases due to the increased risk of aggregation. However, in this study, the negative charge of CMCS (Zeta potential = −30.08 mV) effectively suppressed particle aggregation, maintaining superparamagnetism in the dispersed state.

The AC of both CMCS-Fe₃O₄ NPs and Fe₃O₄ NPs demonstrated a concentration-dependent enhancement, exhibiting a positive correlation with increasing initial manganese ion concentrations. Moreover, the AC of CMCS-Fe₃O₄ NPs was consistently higher as against Fe₃O₄ NPs. From a medical mechanism perspective, the abundant carboxyl and amino functional groups in CMCS molecules can chelate manganese ions to form stable complexes, thereby enhancing the adsorption capacity of the NPs for manganese ions. In biological systems, this adsorption effect contributes to the clearance of excess manganese ions, mitigating their toxic impact on neuronal cells and other tissues [19]. Although the coating of CMCS on the surface of Fe₃O₄ NPs has not been experimentally confirmed to increase the specific surface area of the NPs (lacking supporting data from tests on loose or porous structures), the functional groups on the CMCS molecular chains provide additional surface active sites, enabling more manganese ions to interact with and be adsorbed by the NPs.The pH of the human physiological environment is typically maintained within a relatively stable range, such as the blood pH of approximately 7.35 −7.45. The NPs’ optimal adsorption performance near the physiological pH suggests that they may effectively adsorb manganese ions in the in vivo environment [20]. From the perspective of the adsorption mechanism, the high efficiency of CMCS-Fe₃O₄ NPs in adsorbing manganese ions is primarily attributed to the synergistic effect of chemical complexation, electrostatic interactions, and physical adsorption. The carboxyl groups (-COOH) on the surface of CMCS can form stable complexes with manganese ions through coordination bonds, as expressed by the reaction: -COOH + Mn²⁺ → -COO-Mn⁺ + H⁺. This chemical complexation is the core mechanism behind the enhanced adsorption capacity. Additionally, when the solution pH approaches 7, the Zeta potential of CMCS-Fe₃O₄ NPs is −30.08 mV, and the surface is negatively charged, which enhances the capture ability of Mn²⁺ through electrostatic attraction. Furthermore, the high specific surface area of Fe₃O₄ NPs provides adsorption sites for manganese ions, while the porous network structure formed after CMCS coating further expands the adsorption interface. Isotherm model fitting revealed that the adsorption of manganese ions by CMCS-Fe₃O₄ NPs fits better with the Langmuir model, with a correlation coefficient (R²) of 0.98, indicating that adsorption predominantly occurs via monolayer chemical adsorption. The maximum theoretical adsorption capacity (q_max) is 125.6 mg/g, which is in close agreement with the experimental saturated adsorption value of 118.3 mg/g. In contrast, the fitting results for the Freundlich and Temkin models were relatively weak, further supporting the chemical adsorption-based mechanism. It is worth noting that when the manganese ion concentration increased from 10 mg/L to 50 mg/L, the adsorption capacity of CMCS-Fe₃O₄ NPs increased from 72.3 mg/g to 118.3 mg/g. This increase can be attributed to the reduced mass transfer resistance and the increased probability of collisions between active sites due to the higher concentration. However, this concentration dependence reflects only the differences in the adsorption rate during the kinetic process. To accurately characterize the material’s saturated adsorption capacity (Q_max), thermodynamic analysis should be combined with longer adsorption times (e.g., 24 h) and higher concentration gradients (e.g., 100–200 mg/L).

Cellular experimental results demonstrated that, as against the BG, the MG exhibited markedly decreased multiplication activity, raised AR, and markedly reduced transfer and attack capabilities, consistent with the clinical phenomenon of manganese-induced neurotoxicity. The toxic effects of manganese ions on nerve cells may involve multiple mechanisms. Manganese ions can disrupt the intracellular redox balance, generating a large amount of ROS and inducing oxidative stress [21]. Oxidative stress can damage biological macromolecules such as cell membranes, proteins, and DNA, affecting normal cellular metabolism and function. On the other hand, manganese ions can also interfere with the synthesis, release, and metabolism of neurotransmitters, disrupting nerve signal transmission and thus affecting the normal function of nerve cells. As against the MG, the IG showed markedly raised multiplication activity, decreased AR, and markedly enhanced transfer and attack capabilities. This indicates that CMCS-Fe₃O₄ NPs can mitigate the damage of manganese ions to nerve cells to some extent. The mechanism may primarily be related to the NPs’ adsorption of manganese ions. CMCS-Fe₃O₄ NPs can chelate with manganese ions through the CMCS functional groups on their surface, removing manganese ions from the cellular environment and thereby reducing their toxicity to nerve cells [22]. Regeneration performance and long-term stability are critical indicators for evaluating the practical application value of adsorption materials, particularly in wastewater treatment, as they directly impact material usage costs and environmental safety. In this study, CMCS-Fe₃O₄ NPs retained over 80% of their adsorption capacity after five adsorption-desorption cycles, outperforming some existing Fe₃O₄-based composite adsorbents (e.g., chitosan-Fe₃O₄NPs retained only 65% adsorption capacity after five cycles). This advantage is attributed to the stable core-shell structure formed by the CMCS coating layer. During desorption, HCl only interacts with the Mn²⁺ adsorbed on the material surface without disrupting the coordination bonds between CMCS and Fe₃O₄, thereby preserving the material’s adsorption activity. Long-term stability tests further confirmed that the CMCS coating effectively inhibits oxidation and leaching of Fe₃O₄ NPs: The hydroxyl and carboxyl groups in CMCS molecules form stable coordination bonds with Fe³⁺, preventing the release of Fe ions from the material core. The hydrophilic nature of CMCS ensures excellent dispersibility of the material in aqueous environments, avoiding structural collapse caused by aggregation. Moreover, the stability of the material in simulated wastewater and physiological saline suggests its applicability not only for manganese ion wastewater treatment but also for potential aquatic bioremediation or in vivo applications. Collectively, the high renderability and long-term stability of CMCS-Fe₃O₄ NPs address the limitations of traditional adsorbents, such as high single-use costs and risks of secondary pollution, significantly enhancing their feasibility for practical wastewater treatment applications and providing experimental evidence for future large-scale implementation. This study further elucidated the mechanism by which CMCS-Fe₃O₄ NPs antagonize manganese-induced neurotoxicity through ROS level detection. The results showed that the relative fluorescence intensity of ROS in the MG(treated with 500 μmol/L MnSO₄ for 24 h) was significantly elevated (318.52 ± 11.36) compared to the BG(100.00 ± 4.89) (P < 0.01), consistent with previous findings that excess manganese ions induce substantial ROS generation in neuronal cells by disrupting the mitochondrial respiratory chain and activating NADPH oxidase. This confirms that manganese exposure causes significant oxidative stress damage in SH-SY5Y cells, with oxidative stress being a core pathological mechanism in manganese-induced neurotoxicity. Notably, after pretreatment with CMCS-Fe₃O₄ NPs, intracellular ROS levels (182.47 ± 7.93) were significantly reduced compared to the MG(P < 0.01). Although still higher than the BG (P < 0.05), a clear alleviation of oxidative stress was demonstrated. This outcome is not coincidental but directly linked to the structural characteristics and mechanistic actions of CMCS-Fe₃O₄ NPs. The abundant carboxyl (-COOH) and hydroxyl (-OH) groups on the CMCS molecular chains act as free radical scavengers, directly capturing excess intracellular ROS (e.g., superoxide anions, hydroxyl radicals), thereby mitigating oxidative damage to lipids, proteins, and nucleic acids. The core-shell structure of CMCS-Fe₃O₄ NPs endows them with excellent manganese ion chelation capacity. The carboxyl groups of CMCS form stable complexes with manganese ions, reducing the concentration of extracellular free manganese ions. This minimizes the disruption of oxidative metabolic systems after manganese ions enter cells, thereby suppressing ROS generation at its source.

The necessity and mechanistic rationale for developing CMCS-Fe₃O₄ NPs in this study are fully supported by the experimental results. From a material design perspective, unmodified Fe₃O₄NPs suffer from poor stability and aggregation tendencies, which limit their adsorption efficiency and in vivo applicability. After CMCS coating, the composite NPs form a distinct core-shell structure (as observed in TEM images) with a more uniform particle size distribution. This structural improvement not only addresses the aggregation issue of Fe₃O₄NPs but also preserves their superparamagnetic properties (as confirmed by VSM results, showing negligible coercivity and remanent magnetization). The superparamagnetism is critical for potential in vivo targeted localization and separation, an advantage unattainable with CMCS alone (which lacks magnetic responsiveness). In terms of manganese ion adsorption, experimental results clearly demonstrate that CMCS-Fe₃O₄ NPs consistently exhibit higher adsorption capacity than Fe₃O₄NPs across varying initial manganese ion concentrations and pH conditions. This superior adsorption performance stems from the synergistic effects between CMCS and Fe₃O₄NPs.CMCS is a derivative of the natural biopolymer chitin through chemical modification (carboxymethylation), retaining the excellent biocompatibility of chitin while introducing additional carboxyl and amino groups that enable specific chelation with manganese ions to form stable complexes. Simultaneously, Fe₃O₄NPs provide a large specific surface area, offering abundant active sites for manganese ion adsorption. Particularly within the physiological pH range [6–8], CMCS-Fe₃O₄ NPs exhibit optimal adsorption capacity, ensuring their potential efficacy in vivo and representing a key advantage over non-biocompatible adsorbents. Regarding neuroprotective effects, experimental data confirm that CMCS-Fe₃O₄ NPs reverse manganese-induced damage in SH-SY5Y cells: compared to the MG, the IG showed enhanced proliferation, reduced apoptosis, and improved migration and invasion capabilities. The core mechanism lies in the adsorption capacity of CMCS-Fe₃O₄ NPs. By chelating manganese ions in the cellular environment, the composite NPs reduce the concentration of free manganese ions that exert toxic effects on neuronal cells, thereby preventing further damage and creating favorable conditions for functional recovery. In contrast, unmodified Fe₃O₄NPs, due to poor biocompatibility, fail to achieve comparable neuroprotective outcomes, while CMCS alone lacks magnetic targeting capability, limiting its adsorption efficiency due to inadequate enrichment at manganese accumulation sites. This study has certain limitations. It solely used the human neuroblastoma cell line SH-SY5Y to investigate the neuroprotective effects of CMCS-Fe₃O₄ NPs, without involving primary neuronal cells or other types of neural cells. Therefore, there are limitations in assessing the in vivo neuroprotective effects. Compared to traditional chelating agents such as diethylenetriamine pentaacetate (DTPA), although CMCS-Fe₃O₄ NPs possess magnetic responsiveness and high manganese ion adsorption capacity, their chelation efficiency and stability in complex in vivo environments have not been directly compared with clinically used chelators like DTPA. Furthermore, data regarding their biodistribution, metabolism, and long-term toxicity in animal models are lacking. Future studies should expand the cell model types and conduct in vitro and in vivo comparative studies on the adsorption performance and biosafety with classic chelating agents, to further clarify their clinical translation potential.

A critical comparative analysis integrating the research data from this study and existing literature demonstrates that this work transcends the common limitation of composite nanomaterials research that prioritizes in vitro adsorption over in vivo functionality. Through systematic experimentation, it is confirmed that CMCS-Fe₃O₄ NPs not only exhibit excellent manganese ion adsorption performance in vitro but also repair manganese-induced neuronal damage at the cellular level. Furthermore, the characterization of the material and elucidation of its mechanisms address the research gap in how composite nanomaterials simultaneously achieve adsorption and neuroprotection. The core conclusion of this study is that CMCS-Fe₃O₄ NPs, through the synergistic effects of CMCS and Fe₃O₄, overcome key defects of existing materials. Their integrated adsorption-protection-targeting characteristics provide a novel material paradigm and experimental basis for the prevention and treatment of manganese-related neurotoxic diseases. Subsequent animal studies are required to validate their in vivo metabolic safety and therapeutic efficacy to facilitate clinical translation. The above conclusions are supported by corresponding figures: Fig. 5 (adsorption capacity at different concentrations) confirms the material’s high-efficiency adsorption of manganese ions, providing evidence for “reducing manganese ion concentration in the cellular environment.” The cellular functional data in Figs. 7 and 8 visually demonstrate the damage repair effects across multiple dimensions, proliferation, apoptosis, migration, and invasion, thereby forming a complete evidence chain for the adsorption-protection mechanism and ensuring conclusions are firmly data-grounded.

Conclusion

This study successfully prepared CMCS-coated magnetite NPs (CMCS-Fe₃O₄ NPs) and systematically validated their core properties and biological effects. In terms of material characteristics, CMCS was identified as a derivative of natural chitin modified by carboxymethylation. The coating formed a stable core–shell structure. Although the particle size increased compared to bare Fe₃O₄ NPs without improved size distribution uniformity, the more negative Zeta potential effectively inhibited aggregation while retaining superparamagnetism, meeting the requirements for targeting applications. Regarding manganese ion adsorption, CMCS-Fe₃O₄ NPs consistently exhibited higher adsorption capacity than Fe₃O₄ NPs and maintained stable adsorption within the physiological pH range. The high adsorption efficiency was attributed to the chelation between carboxyl groups of CMCS and manganese ions, as well as the abundant active sites provided by Fe₃O₄ NPs. In terms of biological protective effects, CMCS-Fe₃O₄ NPs significantly reversed manganese-induced damage in SH-SY5Y cells: they not only enhanced the proliferative activity of injured cells, reduced apoptosis, and restored migration and invasion capabilities, but also effectively scavenged excess intracellular ROS. These results demonstrate their dual functionality in manganese adsorption and oxidative stress mitigation, offering a novel candidate material for the prevention and treatment of manganese-related neurotoxic diseases.

Future studies should expand the range of cell models by incorporating primary neural cells to validate the universal protective efficacy of the material across neural cells of different origins. Animal experiments should be conducted to systematically investigate the in vivo metabolic pathways, targeted accumulation efficiency, and long-term biosafety of CMCS-Fe₃O₄ NPs. Comparative studies with clinically established chelating agents are also warranted to clarify their advantages and positioning in the treatment of manganese intoxication, thereby laying a critical foundation for translating this material from basic research to clinical application.

Author contributions

XW, QT, ML, and XM designed the study and wrote the manuscript, data and helped to draft the manuscript. All authors have read and approved the final manuscript.

Data availability

All data generated or analyzed during this study are included in this published article.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.