Original Article

The effects of Fe2O3 nanoparticles on catalytic function of human acetylcholinesterase: size and concentration role

Abstract

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Introduction:

Fe₂O₃ NPs can enter cells quickly, pass through the blood-brain barrier and interact with macromolecules. These materials are widely used in different fields, so their risk assessment is among the most critical issues. Acetylcholinesterase (AChE) is a cholinergic enzyme in central and peripheral nervous systems.

Methods:

In this work, the possible effects of Fe₂O₃ NPs on the structure and catalytic activity of AChE were investigated using circular dichroism (CD), surface plasmon resonance (SPR), and fluorescence spectroscopies.

Results:

The outcomes demonstrated that 5 nm Fe₂O₃ NPs inhibit AChE activity through mixed mechanism. While 50 nm Fe₂O₃ NPs caused an enhancement in the catalytic activity up to 60 nM. However, higher concentrations of Fe₂O₃ NPs (above 60 nM) hindered the enzyme activity via mixed mechanism. Fluorescence analysis showed that NPs can quench the fluorescence intensity of AChE that refer to conformational changes. Furthermore, CD results showed that Fe₂O₃ NPs can reduce the α-helix and β-sheet contents of the enzyme and decrease the stability of AChE. Also, the SPR data analysis showed that the affinity between AChE and Fe₂O₃ NPs decreased with rising temperature. After treatment with Fe₂O₃ NPs, the catalytic activity of AChE was assessed in HepG2 cell lines, and the results confirmed the inhibitory effects of Fe₂O₃ NPs on AChE activity in vivo.

Conclusion:

These findings provide helpful information about the impact of Fe₂O₃ NPs on the structure and function of AChE and could offer new insights into the risk assessment of the medical application of nanoparticles.

Keywords: Acetylcholinesterase, Fe2O3 nanoparticles, Fluorescence spectroscopy, Circular dichroism

Introduction

In recent years, the use of various nanoparticles (NPs) has expanded widely in different areas, including medicine, engineering, catalysis, and environmental remediation.¹ This is due to the unique properties of these materials, such as their electrical, optical, chemical, magnetic, and magneto-optical properties.^2-4 Given the widespread use of NPs, risk assessment of these materials is critical, and their toxicity has become one of the main challenges among researchers. So far, different kinds of NPs have been synthesized and developed. Iron oxide NPs (which consist of maghemite (γ-Fe₂O₃) and/or magnetite (Fe₃O₄) particles) are among the most important nanomaterials.⁵

Fe₂O₃ NPs have gained considerable attention because of their unique intrinsic features, such as outstanding biocompatibility and superior magnetic properties.^6,7 These NPs are widely used in a variety of fields, such as sensing technologies, memory storage devices, magnetic separation, magnetic labeling, catalytic processes, and biomedicine (heating for hyperthermia treatments, providing contrast effects for magnetic imaging, and remotely controlling the delivery of targeted drugs).^8-10

NPs are small particles with large surface-to-volume ratios.^11,12 It has been accepted that the small size of NPs can cause these materials to enter cells quickly, pass through the blood-brain barrier, and interact with different kinds of proteins and enzymes.¹³ In general, the conformation and function of enzymes and proteins are widely associated with their tertiary structure and protein dysfunction can lead to various diseases and disorders.¹⁴

Acetylcholinesterase (AChE), a critical serine hydrolase, is secreted into the synaptic space by postsynaptic cholinergic neurons.¹⁵ AChE is a crucial enzyme for the growth and operation of the central nervous system. This enzyme also has a significant impact on neurodevelopment and hematological differentiation.^16,17 In the synaptic space, it promotes the breakdown of acetylcholine into choline and acetate.¹⁸ According to studies, the inhibition of AChE leads to an accumulation of acetylcholine in the synaptic cleft. It disrupts the neurotransmitter levels in the synapses, which overexcites nicotinic and muscarinic acetylcholine receptors and impairs neurotransmission.^19,20

According to the previous experiment, Fe₂O₃ NPs could change brain proteome and affect the cholinergic function of a rat's brain.²¹ Therefore, the main objective of the present work was to investigate the possible effects of Fe₂O₃ NPs on the structure and catalytic activity of AChE via spectroscopic methods. On the other hand, the activity of AChE extracted from HepG2 cell lines was evaluated after exposure to Fe₂O₃ NPs with two different sizes (5 and 50 nm).

Materials and Methods

Materials

Human acetylcholinesterase enzyme (AChE), 5,5’-dithio-bis-(2-nitrobenzoic) acid (DTNB), acetylthiocholine iodide ethanaminium, and Fe₂O₃ NPs (Fe₂O₄ NPs; with the size of 5 and 50 nm), N-ethyl-N-(3-dimethyl aminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), NaCl, and NaOH were provided from Sigma Aldrich Company (St. Louis, MO, USA).

Methods

Preparation and characterization of NPs

Fe₂O₃ NPs were prepared from Sigma-Aldrich Company and dispersed by sonication (10 min, 750 W, and 20 kHz) in phosphate buffer (pH 7) before use. Size distribution and NPs dispersion were evaluated by the dynamic light scattering (DLS) and TEM methods. Fig. 1 shows Fe₂O₃ NP in crystalline phase with 5 and 50 nm sizes. In this work, the high-purity Fe₂O₃ NPs (99%) were used without coating. The prepared particles had γ-Fe₂O₃ crystalline phase with spherical morphology. Also, the specific surface areas of Fe₂O₃ NP with 5 nm and 50 nm were 450–2000 m²/g and 50–245 m2/g, respectively.

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Fig. 1.

DLS analysis of Fe₂O₃ NPs with 5 nm (A) and 50 nm (B) in size.

Fig. 1.

DLS analysis of Fe₂O₃ NPs with 5 nm (A) and 50 nm (B) in size.

AChE activity assay

The catalytic activity of AChE in the presence of Fe₂O₃ NP was investigated using Ellman’s colorimetric method.²² For this purpose, additional concentrations of Fe₂O₃ NP (0-2500 nM) were added to the reaction solution containing phosphate buffer (0.1 M, pH 7.0) and AChE (0.7 μg/mL). After incubating for 2 hours, DTNB and acetylcholine iodide (as a substrate) were added to the mixture. DTNB and substrate had final concentrations of 0.33 mM and 1.56 mM, respectively. Following incubation of the prepared reaction mixture for 5.0 minutes at 25 °C, the rate of acetylcholine iodide hydrolysis and the formation of 5-thio-2-nitrobenzoate were measured spectrophotometrically using a UV-visible spectrometer (T-60, PG Instruments LTD., Leicestershire, UK) at 412 nm. AChE decomposes acetylcholine iodide and produces thiocholine. The interaction between thiocholine and DTNB results in 5-thio-2-nitrobenzoate production.^23,24

Circular dichroism spectroscopy

To obtain insight into the potential effects of Fe₂O₃ NPs on the secondary structure of AChE, the circular dichroism spectroscopy(CD) spectra of AChE in the absence or presence of Fe₂O₃ NPs were measured in the far UV spectral region (200-250 nm) using a Jasco model spectropolarimeter at 25 °C. In this regard, different concentrations of Fe₂O₃ NPs (0-1000 nM) were added to 1.0 mL of 20 mM phosphate buffer solution (pH 7.4; 310 K) containing 2.0 mg/mL AChE. The prepared reaction mixtures were incubated for 5 minutes, and the far UV spectra of the samples were recorded from 200 nm to 250 nm.²⁵ Finally, CDNN software was used to calculate the percentage of changes in the secondary structural elements of the enzyme.

Surface plasmon resonance (SPR) measurements

The kinetic parameters of the AChE-Fe₂O₃ NPs interaction were investigated using SPR analysis at four different temperatures (298, 303, 310, and 313 K) to obtain the rate constants and affinity between AChE and Fe₂O₃ NPs. All the SPR analysis was carried out on a double-circuit channel MP-SPR Navi^TM 210A device with gold chips (Bio Navis Ltd. Tampere, Finland) after immobilizing AChE on the carboxymethyl dextran (CMD) Au chip. In this regard, the CMD sensor chip was washed using a 10 mM acetate buffer solution (pH=4.5) and then appended to the SPR device. Subsequently, to establish a stable baseline, the chip surface was cleaned by injecting NaCl (2 M) and NaOH (0.1 M) into the apparatus for 30 min at a circuit rate of 30 μL/min. To activate the chip surface, a solution containing 0.05 M NHS and 0.2 M EDC was injected into the device for 7 min. Then, the prepared AChE solution was introduced into channel 1. Channel 2 was used as a reference channel. Finally, the immobilization process was completed by using 0.1 M ethanolamine-HCl (pH=8.5), which was injected into the Au chip surface to block the non-specific binding sites. In addition, to investigate the binding of Fe₂O₃ NPs to the immobilized AChE and the analysis of kinetic and thermodynamic parameters, additional concentrations of 5 nm Fe₂O₃ NPs (5, 100, 200, 400, and 800 nM) and 50 nm Fe₂O₃ NPs (0.5, 1, 2, 4, and 8 µM) were injected into channel 1. SPR Navi^TM data viewer software and Trace Drawer^TM were utilized for data analysis and the calculation of the interaction parameters, respectively.

Fluorescence spectroscopy

In this work, fluorescence spectroscopy was used to evaluate the conformational changes of AChE upon interaction with Fe₂O₃ NPs. For this purpose, the intrinsic fluorescence intensity of AChE was measured without and with various concentrations of Fe₂O₃ NPs (50-1000 nM) using a spectrofluorometer (Jasco, FP-750, Kyoto, Japan) with a 1.0 cm quartz cuvette. A fixed concentration of AChE (0.5 mg/mL) was incubated with different dosages of Fe₂O₃ NPs (50-1000 nM) for 5 minutes at 310 K in phosphate buffer solution (pH 7.4). Then, the enzyme was excited at 280 nm, and the emission spectra of the samples were recorded in the range of 300-500 nm. The slit width for the excitation and emission was 5 nm.¹⁸

Cell culture

To examine the potential effects of Fe₂O₃ NPs (5 nm and 50 nm) on the catalytic activity of AChE, HepG2 cell lines were cultivated in Williams-fetal bovine serum (Williams-FBS) media containing 10% FBS, 100 U/mL ampicillin, and 100 g/mL streptomycin. The cells were seeded per well of a 12-well culture plate and treated with a 2 µM concentration of Fe₂O₃ NPs. The treated and untreated control cells were incubated at 5% CO₂ in a humidified atmosphere of 95% air and 37 °C for 24 hours. Then, the inhibitory effect of Fe₂O₃ nanoparticles on AChE activity was evaluated by preparing cell lysate.²⁶ The experiments were repeated three times.

Statistical evaluation

All of the experimental data were analyzed by version 11 of SPSS software, and the expression of the data was done as mean ± standard deviation (SD). One-way variance (ANOVA) was used for the statistical analysis, followed by a multiple-range test using Dennett's approach. Differences at P < 0.05 were considered significant results.

Results and Discussion

Because it may regulate the cholinergic neurotransmitter in the synaptic cleft, AChE is a crucial enzyme for the nervous system's proper operation. Therefore, it is one of the main target enzymes in neural toxicity and progressive neurological disorders such as Alzheimer's. Fe₂O₃ NPs are applied in various scientific fields and different industries.²⁷ Applications of these NPs in different sizes have been rising in recent years. Despite the wide range of applications of NPs, particularly in medicine, their neurotoxicity poses challenges. Therefore, investigating the toxicity of NPs on various macromolecules, such as enzymes, proteins, and nucleic acids, is of great importance.

Effects of Fe₂ O₃ NPs on catalytic activity of AChE

Due to their unique physiochemical properties, Fe₂O₃ NPs are widely used in many in vivo and in vitro research projects.²⁸ These materials are one of the most significant nanomaterials because of their extensive use in magnetic resonance imaging (MRI), ultrasound, optical imaging, X-ray imaging, drug delivery, gene delivery, etc.^29-31 Previous studies confirmed that this type of nanomaterials causes harsh oxidative damage in living systems.³² By considering previous results and physiological signs of Fe₂O₃ NPs poisoning, the main objective of this study was to study the possible effects of Fe₂O₃ NPs in two different sizes, 5 nm and 50 nm, on the structure and catalytic activity of AChE. As shown in Fig. 2A, the Fe₂O₃ NPs with a 5 nm size inhibited the enzyme activity in a dose-dependent manner. AChE's catalytic activity was shown to be affected in two ways by 50 nm Fe₂O₃ NPs (Fig. 2B), and a continuous decrease of enzyme activity was observed with increasing concentrations of 5 nm Fe₂O₃ NPs. The results indicated that the enzyme's activity increased significantly with increasing the Fe₂O₃ NPs concentration (up to 60 nM). However, at higher concentrations than 60 nM, a reduction in enzyme activity was observed. It can be concluded that the particles of large size interfere with or compete at the catalytic site of the enzymes and inhibit their action. This suggests that competition with the substrate due to the hydrophobicity of the particle and NPs forming micelles with the microsomal membrane leads to a change in membrane integrity and, thus, enzyme inactivation.³³ In addition, with increasing the size of nanoparticles, their entrance into the active site of the enzymes reduces. Therefore, the conformation of the active site cannot be influenced by particles of large size.³⁴

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Fig. 2.

AChE catalytic activity in the presence of Fe₂O₃ NPs of size 5 nm (A) and 50 nm ‎‎(B) at 25 °C. Data was shown as mean±SD.‎

Fig. 2.

AChE catalytic activity in the presence of Fe₂O₃ NPs of size 5 nm (A) and 50 nm ‎‎(B) at 25 °C. Data was shown as mean±SD.‎

In addition, the inhibition type of the Fe₂O₃ NPs (5 and 50 nm) in the AChE action was evaluated. In this regard, AChE was incubated with different concentrations of 5 nm Fe₂O₃ NPs (0, 60, and 150 nM) and 50 nm Fe₂O₃ NPs (0, 700, and 1000 nM) and then the activity of the AChE was recorded spectrophotometrically. The results indicated that the catalytic activity of AChE was inhibited in the presence of increasing concentrations of Fe₂O₃ NPs through a mixed-type mechanism (Fig. 3). The calculated K_m and V_max values are listed in Table 1. As shown in Fig. 3A and B, increasing the 5 nm Fe₂O₃ NPs concentration mainly increased the K_mvalue from 1.81±0.09 mM for the free enzyme to 2.43±0.11 mM for the AChE-Fe₂O₃ NPs complex. Also, the V_max values decreased from 793.15±56.87 nmol/min for the free AChE to 364.28±23.65 nmol/min for the AChE upon interaction with Fe₂O₃ NPs. On the other hand, 50 nm Fe₂O₃ NPs caused a reduction in the K_m (from 1.81±0.09 mM to 1.31±0.05 mM) and V_max (from 793.15±56.87 nmol/min to 207.17±13.21 nmol/min) values of AChE (Fig. 3C and D). According to these results and based on the data reported in Table 1, it was concluded that Fe₂O₃ NPs (5 nm and 50 nm) are able to inhibit the catalytic activity of AChE through mixed mechanism of inhibition.³⁵ According to the results 5 nm NPs could bind to the free enzyme more than enzyme-substrate complex that refer to a competitive-noncompetitive inhibition while the 50 nm NPs prefer to bind to the enzyme-substrate complex (noncompetitive-uncompetitive type of inhibition).

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Fig. 3.

Michaelis-Menten and Lineweaver-Burk plots of AChE with and without various concentrations of Fe₂O₃ NPs with 5 nm (A and B) and 50 nm (C and D) in size.

Fig. 3.

Michaelis-Menten and Lineweaver-Burk plots of AChE with and without various concentrations of Fe₂O₃ NPs with 5 nm (A and B) and 50 nm (C and D) in size.

Fluorescence spectroscopy studies

Fluorescence spectroscopy is one of the most simple, sensitive, and inexpensive methods for evaluating the interactions between macromolecules and ligands, such as protein-ligand interactions. Three different aromatic amino acid residues, including tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe), exist in the AChE structure.³⁶ The intrinsic fluorescence intensity of AChE comes from these residues, which are very sensitive to rearrangements in the polarity of their environment.¹⁸ In this work, the effect of additional concentrations of Fe₂O₃ NPs on the emission intensity of AChE was investigated. The maximal emission spectrum of the AChE was observed at 360 nm, a characteristic of Trp residues in a slightly hydrophilic environment. The results showed that Fe₂O₃ NPs can reduce an enzyme's intrinsic emission by changing its conformation and the polar micro-region of aromatic amino acids (Fig. 4). The fluorescence quenching effects of AChE in the presence of Fe₂O₃ NPs depict a less compact structure due to increased distances between the fluorophore molecules as well as their more significant interactions with the hydrophilic environment that result in fluorescence quenching.³⁶ The study also showed no shift in the position of the Trp residues, which means that although the inhibitor interacted very closely with AChE to quench fluorescence, the secondary structures of the enzyme may not have been altered.³⁷ It is suggested that a new non-fluorescent complex was formed between AChE and Fe₂O₃ NPs. The tertiary structure of AChE, in particular the substrate entrance gate of the active site, may be impacted by these particles, which inhibits the enzyme's catalytic activity.

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Fig. 4.

Fluorescence spectra of AChE without and with different concentrations (50, 500, and 1000 nM) of Fe₂O₃ NPs (5 nm, and 50 nm).

Fig. 4.

Fluorescence spectra of AChE without and with different concentrations (50, 500, and 1000 nM) of Fe₂O₃ NPs (5 nm, and 50 nm).

CD spectroscopy studies

CD spectroscopy is one of the acceptable methods for monitoring the conformational and structural alternations in the secondary structure of different macromolecules, such as proteins induced via binding to specific molecular substances.³⁸ To look into potential impacts on the secondary structure of AChE, a specific concentration (2 mg/mL) of AChE was incubated with various concentrations of Fe₂O₃ NPs (50–1000 nM) for 3 min, and then, the CD spectra of the samples were recorded in the range of 200-250 nm. In general, the CD spectra of AChE show two main negative bonds, which are located at 208 nm and 222 nm.³⁹ These negative bands are related to π ⟶ π* and n ⟶ π* transitions of amide groups in α-helical structure, respectively. Also, a negative band around 217 nm corresponds to the β-sheets.¹⁸ Fig. 5 shows the CD spectra of AChE upon interaction with Fe₂O₃ NPs. As shown in this figure, after incubating with Fe₂O₃ NPs, the α-helix and β-sheets contents decreased in the AChE structure. According to Table 2, the native AChE showed a preponderance of 30.1% α-helix, 21.9% β-sheets, 15.2 % β-turn, and 32.8% unordered structures. However, the percentages of these elements changed upon interaction with Fe₂O₃ NPs of different sizes. The results indicated that the contents of α-helix were decreased to 18.3% and 10.3% in the presence of Fe₂O₃ NPs with 5 nm and 50 nm (1000 nM), respectively. In addition, Fe₂O₃ NPs with 5 nm and 50 nm reduced the percentages of β-sheets to 13.8% and 8.5%, respectively. So, these results showed that Fe₂O₃ NPs can reduce the stability of AChE by decreasing the α-helical structures that refer to the denaturing of proteins in the presence of NPs. The outcomes indicated the alterations in conformation and secondary structure of AChE in the presence of Fe₂O₃ NPs with a 50 nm size were higher than those of 5 nm Fe₂O₃ NPs. Due to the existence of the active site of AChE between α-helix and β-sheet domains, it can be concluded that the catalytic activity of the enzyme can be influenced by any changes in their conformations and contents.¹⁸ The structure of the active site may also be impacted by AChE's secondary conformational changes, which impact the substrate entrance gate and enzyme activity.⁴⁰ Therefore, it is suggested that the active site rearrangement caused by Fe₂O₃ NPs may eventually result in substrate traffic at the entrance gate.

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Fig. 5.

CD spectra of AChE in the absence and presence of additional concentrations (50, 500, and ‎‎1000 nM) of Fe₂O₃ NPs.‎

Fig. 5.

CD spectra of AChE in the absence and presence of additional concentrations (50, 500, and ‎‎1000 nM) of Fe₂O₃ NPs.‎

SPR results

The kinetic parameters of the interaction of Fe₂O₃ NPs with immobilized AChE were assessed using the SPR technique. These parameters reveal the affinity between a ligand and a macromolecule. To this end, different concentrations of Fe₂O₃ NPs were injected into the chip surface, and then the SPR signals were recorded. Fig. 6 shows the SPR sensorgram of the binding of AChE with Fe₂O₃ NPs at 298K (the SPR sensorgrams at 303, 310, and 313 K are not shown). It is clear that with increasing the concentration of Fe₂O₃ NPs the binding signal gradually increased, too. The equilibrium constant (K_D) values were calculated, and the findings are summarized in Table 3. According to this table, the low values of K_Dindicate a high affinity between ligands and AChE.⁴¹ Additionally, the findings showed that the K_D values increased as the temperature rose, supporting that the affinity between AChE and Fe₂O₃ NPs and the reaction rate decreased with rising temperature.⁴²

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Fig. 6.

Dose-response sensorgrams of AChE in the presence of Fe₂O₃ NPs with 5 nm (A) and 50 nm (B) in size at 298 K.

Fig. 6.

Dose-response sensorgrams of AChE in the presence of Fe₂O₃ NPs with 5 nm (A) and 50 nm (B) in size at 298 K.

Thermodynamic analysis

It has been reported that various acting forces, such as hydrogen bonds, electrostatic forces, Vander Waals forces, and hydrophobic interactions, play a critical role in the complex formation between a ligand and a macromolecule.^38,43 In the present work, the thermodynamic parameters were calculated using equations 1 and 2.

Here, R (8.314 J/mol/K) is the universal gas constant and T denotes the absolute temperature, respectively.

The plot of lnK_D against 1/T (Van’t Hoff plot) was constructed and then the slope and the intercept of the plot was used for the calculation of ΔH and ΔS, respectively (Fig. 7). The calculated values are reported in Table 3. Based on these data, it can be concluded that Fe₂O₃ NPs bind AChE non-spontaneously (ΔG>0). Also, the positive values of ΔH means that the system has gotten energy from the surroundings in the form of heat. In addition, the reaction was an endothermic since the products have a greater energy level than the reactants and the net heat was absorbed. A positive ΔH and negative ΔS cause a positive ΔG so the reaction is not spontaneous.

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Fig. 7.

Van't Hoff curves for the binding of Fe₂O₃ NPs with 5 nm (A) and 50 nm (B) in ‎size to AChE.‎

Fig. 7.

Van't Hoff curves for the binding of Fe₂O₃ NPs with 5 nm (A) and 50 nm (B) in ‎size to AChE.‎

AChE activity of HepG2 cells

In this work, HepG2 cells were utilized as a model to investigate the impact of Fe₂O₃ NPs on AChEcatalyticactivity inside the cells. For this purpose, the prepared cell lysates were used to assess the catalytic activity of AChE. The findings are shown in Fig. 8. It can be seen from this figure that Fe₂O₃ NPs caused a decrease in AChE activity. However, the inhibitory effect of Fe₂O₃ NPs with a 5 nm size (65 %) was significantly higher than that of a 50 nm size (20%). Therefore, it can be concluded that Fe₂O₃ NPs inhibit the catalyticactivity of AChEin a size-dependent manner. Based on these results, nanoparticles with a smaller size can easily cross the blood-brain barrier, interact with various neurological targets such as AChE, and cause neurotoxic effects.⁴⁴ According to this study, due to their interaction with AChE, Fe₂O₃ NPs (of various sizes) may not be safe or even be neurotoxic. However, the small particles have a more significant neurotoxic effect on the native structure and catalyticactivity of AChE.^44,45

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Fig. 8.

AChE activity assessment in HepG2 cell lines in the absence and presence of Fe₂O₃ ‎NPs. Data was shown as mean±SD and star symbols show significant difference (P<0.05) in ‎comparison with the control (without NPs). ‎

Fig. 8.

AChE activity assessment in HepG2 cell lines in the absence and presence of Fe₂O₃ ‎NPs. Data was shown as mean±SD and star symbols show significant difference (P<0.05) in ‎comparison with the control (without NPs). ‎

Conclusion

In this work, the inhibitory effects of Fe₂O₃ NPs (with two different sizes, 5 nm, and 50 nm) on the catalytic activity of AChE were investigated, and the obtained results indicated that Fe₂O₃ NPs can inhibit AChE activity in a dose-dependent manner. The results confirmed the inhibitory effect of 5 nm Fe₂O₃ NPs on AChE activity via mixed mechanism. However, it was observed that the AChE activity was increased in the presence of 50 nm Fe₂O₃ NPs (up to 60 nM) and then decreased. Also, conformational studies were performed using fluorescence and CD spectroscopy analysis. The results suggested that Fe₂O₃ NPs could change the secondary and tertiary structures of the enzyme by reducing the amount of α-helix and β-sheet, leading to the unfolding of the enzyme structure. According to the results, the conformational changes of the enzyme in the presence of 50 nm Fe₂O₃ NPs were higher than those of 5 nm Fe₂O₃ NPs. On the other hand, the interaction between AChE and Fe₂O₃ NPs was studied using the SPR method, and the results indicated that the K_D values increased with rising temperature, suggesting a reduction in the affinity of AChE towards Fe₂O₃ NPs. Thermodynamic studies revealed that the AChE/Fe₂O₃ NPs complex was formed through a nonspontaneous process. In addition, the effect of Fe₂O₃ NPs on AChEcatalyticactivity was evaluated in HepG2 cell lines, and the obtained results showed that the inhibitory effect of Fe₂O₃ NPs with a 5 nm size was significantly higher than that of a 50 nm size, possibly due to increased surface area to volume ratio. This study provided new insights into the impact of Fe₂O₃ NPs on the function and structure of AChE, and a novel mechanism of Fe₂O₃ NPs poisoning in the dysfunction of the cholinergic system. Considering the increasing application of NPs in different fields and specific features of NPs such as high reactivity, penetration through the blood-brain barrier, and oxidative damage, our results provide strong reasons why the application of NPs should be limited in all aspects of life. It is valuable to notice that the results of this study could help extend the knowledge of utilizing Fe₂O₃ NPs for poisoning in the neural system.

Competing Interests

We wish to confirm that there are no known conflicts of interest associated with this publication.

Ethical Statement

None to be stated.

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