Impact of particle size and surface modifications on the neurotoxic potential of copper oxide nanoparticles

Copper (Cu), a vital trace element, is involved in several physiological functions, such as neurotransmission, energy metabolism, and antioxidant defense¹. The health hazard of Cu toxicity in the general population is minimal as its homeostasis is regulated at physiological levels. In the last two decades, CuO nanoparticles (NPs) have been widely applied in various fields, such as electronics, energy, and healthcare owing to their high surface area-to-volume ratio, catalytic activity, and anti-bacterial capabilities². The potential adverse effects of these NPs are serious health concerns, necessitating thorough toxicological evaluations. Previous studies have revealed that CuO NPs may enter the human body via ingestion, inhalation, or dermal route and can potentially accumulate in various organs³. The potential targets for CuO NP toxicity include the lungs, kidneys, and liver⁴. CuO NPs are reported to cross the blood–brain barrier (BBB). This excessive Cu exposure can lead to neurotoxicity characterized by cognitive impairment, motor dysfunction, and neurodegenerative disorders (NDs)^5,6,7.

Previous in vitro and cell studies have demonstrated that CuO NPs exert toxic effects on primary brain cells^8,9. Analysis of adhesion kinetics, growth, proliferation, and DNA damage revealed that CuO NPs exert cytotoxic and genotoxic effects on the primary cultures of brain microvascular endothelial cells and astrocytes¹⁰. CuO NPs exhibit anti-proliferative properties and can induce cell death in glioma and neural cells^11,12. One study demonstrated that CuO NPs promote apoptosis in glial and neuronal cell lines¹³. At subtoxic doses, CuO NPs activate the NF-κB signaling pathway and enhance amyloid precursor protein expression in neural cells, suggesting a correlation between NPs and NDs¹⁴.

In vivo studies have also reported the neurotoxic effects of CuO NPs. For example, mice intranasally exposed to CuO NPs exhibited nerve cell damage and dysfunction in the cerebellum, cerebral cortex, hippocampus, and striatum¹⁵. Additionally, CuO NPs altered the metabolic and antioxidant characteristics of brain tissues, acetylcholinesterase (AChE) activities, glutathione levels, and lipid peroxidation and downregulated the expression of the cytochrome P-450 enzyme system^16,17,18. In higher organisms, AChE is involved in neurotransmission, cognition, and memory. The principal function of AChE in cholinergic synapses is to hydrolyze the neurotransmitter acetylcholine. However, ingested NPs may bind to AChE and alter its activity¹⁹ .

NPs promote brain tissue damage by inducing apoptosis, inflammation, protein aggregation, and oxidative stress. These mechanisms can lead to the onset and progression of degenerative diseases. Limited studies have examined the correlation between CuO NPs and specific disorders. Therefore, there is a need to evaluate the health risks of CuO NPs, especially ND risk.

This study aimed to examine the neurotoxicity of CuO NPs and assess the effects of their size, surface coating, and released Cu²⁺ ions on the neurotoxic effects. CuO NPs of varying sizes, as well as particles of similar dimensions with or without surface coating, were synthesized. This approach enables the comprehensive evaluation of the effect of particle size and surface coating on neurotoxicity. The polymer polyvinylpyrrolidone (PVP) was selected to examine the effect of coating on the neurotoxicity of CuO NPs, ensuring comparability with other research endeavors. PC-12 cells, which are routinely used to examine neurotoxic effects and can differentiate into neuron-like cells, were used as the in vitro model.

Additionally, this study examined the impact of CuO NPs on the expression of genes associated with the dopaminergic system (Th, Maoa, and Comt) and neurodegeneration initiation (Snca, Prkn, and Gpr37) and AChE enzyme activity.

NP Synthesis and characterization

CuO NPs were synthesized using the precipitation method with cupric nitrate and copper sulfate as precursors. First, 0.5 M aqueous solutions of cupric nitrate and copper sulfate were prepared. The aqueous cupric nitrate and copper sulfate solutions were incubated with 0.5 M aqueous sodium hydroxide solution (added dropwise) with constant vigorous stirring at room temperature to obtain black residue (pH 10). The precipitate was centrifuged, washed with deionized water (to neutralize pH), and divided into two portions. The first portion was subjected to calcination at 80 °C, while the second portion was annealed at 400 °C for 2 h to obtain the black powder, which was finely ground and stored in a vacuum desiccator.

PVP-coated CuO NPs were synthesized by combining the capping agent (PVP) and precursor salts (cupric nitrate) in a 1:1 ratio with the precursor salt (copper acetate monohydrate). The CuO NPs were coated separately with PVP during synthesis.

The particle shape and size were analyzed using a field-emission scanning electron microscope (FEI Nova NanoSEM 450). The mean particle diameter was estimated by analyzing 100 particles (ImageJ, National Institutes of Health). The elements were screened using an energy-dispersive detector (Bruker XFlash 6I30). The CuO NPs were ultrasonicated for 5 min in deionized water at 1 mg/mL and analyzed using a dynamic light scattering instrument (DLS, Sympatec Nanophox) to determine the hydrodynamic diameter. The zeta potential was calculated using Beckman Coulter (Delsa™ Nano). Fourier-transform infrared (FTIR) spectroscopy was used to determine the properties of the functional groups at wavelengths of 400–4000 cm⁻¹ using an FTIR spectrometer (Bruker Tensor-27).

A solid-state ultraviolet–visible (UV–Vis) spectrophotometer (Jasco) was used to analyze the optical characteristics of CuO in the 200–800 nm region. The absorption edge of Tauc plot was extrapolated from the UV–vis spectra to determine the optical bandgap of NPs.

Cell culture and NP treatment

PC-12 (rat pheochromocytoma) cells, which were obtained from the National Centre for Cell Science (Pune, India), were maintained in F-12 Ham, Kaighn’s modification medium supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, and an antibiotic solution containing 10,000U of penicillin and 10 mg of streptomycin at 37 °C in a humidified 5% CO₂ incubator until 80%–90% confluency.

Before NP treatment, an optimization study was performed to establish the appropriate cell density (2000 to 5 × 10⁴ cells per well). This optimization study aimed to mitigate cell death from nutrient depletion in the culture medium during the 96-h treatment. The optimal cell density was determined to be 1 × 10⁴ cells per well, which minimized the potential confounding factors associated with cell stress and nutrient depletion during prolonged NP exposure.

All NPs were sterilized through autoclaving before treatment and tested for contaminants. NPs were dispersed in the complete growth media and subjected to probe sonication for 15 min before each assay. Adhered cells were incubated with various concentrations (0.1, 1, 10, 50, and 100 µg/mL) of NPs for 24–96 h.

Cell viability assay

The viability of cells treated with CuO NPs (0.1–100 µg/ml) for 24–96 h was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The absorbance of the samples at 570 nm was examined using a microplate reader (Biotech Instruments, USA). Cells not treated with NPs served as a control with 100% cell viability. To eliminate NPs interference with the MTT reagent, a blank control was prepared with each concentration of NPs in complete growth media.

The cells were subjected to neutral red uptake (NRU) assay, which was performed using protocols similar to those of the MTT assay, after a specified exposure time, following the manufacturer’s instructions (Neutral Red Cell Assay Kit, Himedia).

Quantification of intracellular Cu²⁺ Ions

The intracellular Cu²⁺ ions (including both internalized nanoparticles and dissociated ions) were quantified by exposing 5 × 10⁴ cells to CuO NPs. Cytotoxicity was first observed at 10 μg/mL after 24 h, and higher concentrations produced a clear dose-dependent increase in toxicity, as shown in Fig. 6. For quantification, cells were exposed to 10 μg/mL CuO NPs for 96 h, washed with phosphate-buffered saline (PBS), trypsinized, and subjected to ultracentrifugation. The resulting cell pellet was digested in nitric acid, and samples were analyzed using atomic absorption spectroscopy (AAS, Shimadzu AA-7000). The AAS values were normalized to the total protein content of each sample, determined by the Bradford assay, to correct for variations in cell number and biomass. Data are expressed as μg Cu per mg protein.

Evaluation of cell membrane integrity

The culture supernatant of cells treated with CuO NPs for different durations (24–96 h) was analyzed using the lactate dehydrogenase (LDH) cell assay kit (Himedia), following the manufacturer’s instructions. The fluorescence intensity was assessed at excitation and emission wavelengths of 560 and 590 nm, respectively.

Evaluation of mitochondrial membrane potential (MMP)

The treated cells were incubated with 1 mM JC-10 dye in the dark for 60 min, following the manufacturer’s instructions. The MMP was determined by quantifying the red-to-green fluorescence intensity ratio.

Evaluation of cellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) production

PC-12 cells were treated with different concentrations of NPs, washed with 100 μL of PBS, and incubated with 100 μL of 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) in the dark for 45 min. The fluorescence intensity was measured using a microplate reader (Ex/Em = 485/535 nm) to determine the ROS levels.

The Griess assay was used to quantify RNS. The culture supernatant of treated cells was subjected to the RNS detection assay with a nitric oxide estimation kit (Himedia), following the manufacturer’s instructions.

Casp3 activation assay

PC-12 cells were incubated with CuO NPs (0.1–100 μg/mL) in 96-well plates for 24 h. The fluorescence intensity (λex = 470, λem = 520 nm) was measured to quantify Casp3 activity using the Caspase-3 assay kit (DEVD-R110 Fluorometric Assay kit, Biotium), following the manufacturer’s instructions.

Determination of pro-inflammatory cytokines levels

The Il6 and Tnf levels were quantified using the enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen, USA), following the manufacturer’s instructions. Briefly, the PC-12 cells were incubated with 0.1–100 μg/mL of CuO NPs for 24 h. ELISA was performed using the cell culture supernatant. The absorbance of the sample at 450 nm was measured using a multi-plate reader.

AChE activity inhibition assay

The effect of CuO NPs on AChE activity was assessed using the acetylcholinesterase inhibitor screening kit (Sigma Aldrich), following the protocol based on the modified Ellman assay. Donepezil (half-maximal inhibitory concentration = 40 nM) served as the positive control.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

RNA was isolated from cells (3 × 10⁶ cells) using Trizol (Invitrogen) and Qiagen TM RNeasy Plus (Qiagen, Valencia, CA), following the manufacturer’s instructions. The isolated RNA was quantified using NanoDrop (NanoDrop Technologies). RNA quality was determined using Bioanalyzer (Agilent Technologies).

Total RNA was isolated from the differentiated PC-12 cells using the RNeasy® Plus Mini kit, following the manufacturer’s instructions. The RNA was reverse-transcribed using random primers (Table S1). qRT-PCR analysis was performed using SYBR green with QuantStudio3 (Applied Biosystems, USA). The expression levels of target genes were normalized to those of Gapdh (internal control).

Effect of released Cu²⁺ ions on cellular toxicity

The concentration of Cu²⁺ions released from the CuO NPs was quantified. The NP sample (10 μg/mL) was incubated in a cell growth medium at 37 °C for 24–96 h. The dispersion was centrifuged at 15,000 rpm for 30 min at 4 °C. Only the supernatant with dissolved Cu²⁺ was used for downstream tests, and not the pellet. The supernatant was collected for quantifying the released ions via AAS. Experiments were performed in triplicate.

Under the same experimental settings, the collected supernatant was subjected to in vitro assays (MTT, DCFDA, IL-6, Casp3 activity, and AChE inhibition) to assess the role of released ions in toxicity and directly compare with the neurotoxicity of CuO NPs.

Statistical analysis

The data are presented as mean ± standard error of mean. The results of the non-treated control group were considered as the control values. The NP characterization data were analyzed using Origin Pro 8.0. (Origin Lab Corporation, MA, USA). Means were compared using two-way analysis of variance, followed Tukey’s multiple comparisons analysis using GraphPad Prism 9.4.1 (USA). All experiments were performed at least thrice.

Characterization of CuO NPs

CuO NPs aggregated in the form of nanoclusters. At high magnification, CuO NPs exhibited a spherical shape (Fig. 1a–c). The mean particle diameter was measured using ImageJ (National Institutes of Health) and represented as histograms (Fig. 1d–f) along with hydrodynamic diameter (Fig. 1g–i). Elemental composition analysis determined using EDX spectroscopy confirmed the presence of Cu and O (Fig. S1). Table 1 shows the particle size, hydrodynamic diameter, and zeta potential of NPs.

The XRD data from JCPDS file 45-–0937 reveals that CuO NPs have a monoclinic crystal structure (Fig. 2a). This is corroborated by the observed diffraction peak positions and relative intensities, which are consistent with the known crystal structure of CuO. Some of the significant peak positions and their corresponding 2θ angles are 35.561° (002), 38.686° (111), 48.840° (202), 53.825° (020), 57.520° (202) and 61.257° (113). In the case of CuO, the (111) plane is the most intense peak in the XRD pattern. The average crystallite sizes for CuO-25, CuO-48, and CuO-P were determined to be 19, 28, and 25 nm, respectively. The variance between the grains and crystallites as well as the particle size determined by FE-SEM were, was in good agreement.

As Cu²⁺ ions are in their crystal lattice, CuO NPs exhibit an absorption peak in the UV region. Cu²⁺ ions in the NP form a shoulder peak in the 550–600 nm region (Fig. 2b). The strength of this peak may increase with a decrease in particle size. According to a recent theoretical paradigm, the correlation between the cellular redox potential and the band gap of metal oxides can explain the oxidative stress-inducing and toxic capabilities of these materials²⁰. The energy difference between the valence band and the conduction band, which is known as the optical band gap (Eg), was determined by extending the linear region of a graph depicting the square of the photon energy (hv)² versus energy. The band gap for CuO-25, CuO-48, and CuO-P, which was calculated using the Tauc plot, was 1.84 eV, 1.25 eV, and 1.24 eV, respectively (Fig. S2).

While FTIR does not provide comprehensive elemental composition or extensive impurity profiles for CuO nanoparticles, it remains an important instrument for determining their surface chemistry. Functional groups and surface-bound species such as hydroxyl, carboxyl, or other organic moieties, which may originate from synthesis precursors or stabilizing agents, can be identified by FTIR, which primarily detects molecular vibrations. FTIR is essential for understanding how surface chemistry affects interactions between nanoparticles and cells in the context of in- vitro investigations²¹.

CuO NP composition and vibration modes were analyzed using FTIR spectroscopy (400–4000 cm⁻¹) (Fig. 3). The vibrations at 480, 530, and 580 cm^–1 are attributed to Cu–O, confirming the purity of CuO NPs. The following two peaks were observed due to the presence of moisture in CuO NPs: 3418 cm⁻¹ (O–H stretching) and 1622 cm⁻¹ (O–H bending). The asymmetric C-O in the CuO structure was connected to the two peaks at 1381 and 1060 cm⁻¹. The PVP coating was the predominant element in the FTIR spectrum of CuO-P NPs. The PVP molecule comprises several functional groups, including carbonyl (C = O) and amide (C-N), exhibiting distinctive absorption peaks (Fig. S3). The broad peak around 3200–3500 cm⁻¹ indicates the O–H stretching of hydroxyl groups in the PVP. The intensity peak around 1638 cm⁻¹ indicates the N–H bending and C-N stretching of amide groups in PVP²². Multiple peaks around 1000–1400 cm⁻¹ correspond to the C-O stretching of the PVP ether groups and the Cu–O stretching of the CuO NPs.

Effect of CuO NPs on cell viability

NPs time-dependently and concentration-dependently decreased cell viability (Fig. 4). In particular, CuO-25 and CuO-48 NPs significantly decreased cell viability at 1 μg/mL with the effect being evident after 72 h of exposure.

Treatment with 10 μg/mL CuO-25 NPs decreased the viability of PC-12 cells to 65% (P < 0.05), which further decreased to 35% (P < 0.05) at 96 h. At a concentration of 50 μg/mL, the viability of CuO-25 NP-treated cells was < 30% (P < 0.05) and 12% (P < 0.01) at 24 and 96 h, respectively. Meanwhile, the viability of cells treated with 100 μg/mL CuO-25 NPs was 17% (P < 0.01) and 6% (P < 0.01) at 24 and 96 h, respectively. The viability of cells treated with 10 μg/mL CuO-48 NPs was 75% (P < 0.05) and 51% (P < 0.05) at 24 and 96 h, respectively. Additionally, the viability of cells treated with 50 μg/mL CuO-48 NPs was 40% (P < 0.05) and 19% (P < 0.01) at 24 and 96 h, respectively, while those treated with 100 μg/mL CuO-48 NPs was 29% (P < 0.05) and 12% (P < 0.01), respectively.

The results of the NRU assay suggested that at 96 h, CuO-25 and CuO-48 NPs decreased the viability of cells to 63% (P < 0.05) and 74% (P < 0.05), respectively (Fig. 5). Treatment with 10 μg/mL CuO-25 NPs significantly decreased cell viability to 60% (P < 0.05) and 25% (P < 0.01) at 24 and 96 h, respectively. The viability of cells treated with 50 μg/mL CuO-25 NPs was 38% (P < 0.05) and 12% (P < 0.01) at 24 and 96 h, respectively, while that of cells treated with 100 μg/mL CuO-25 NPs was 25% (P < 0.01) and 8% (P < 0.01), respectively. At 24 h, the viability of cells treated with 10, 50, and 100 μg/mL CuO-48 NPs was 68% (P < 0.05), 49% (P < 0.05), and 37% (P < 0.05), respectively. Additionally, the viability of cells treated with 10, 50, and 100 μg/mL CuO-48 NPs for 96 h was 34% (P < 0.05), 20% (P < 0.01), and 14% (P < 0.01), respectively.

Comparative analysis of the MTT and NRU assay results suggested that the cytotoxicity of CuO-25 NPs against PC-12 cells was significantly higher than that of CuO-48 NPs at concentrations ≥ 1 μg/mL.

In the CuO-P NP-treated groups, the viability of cells treated with 10 μg/mL CuO-P NPs for 96 h was 55% and 68% in the NRU and MTT assays, respectively. At 100 μg/mL, CuO-P NPs decreased the cell viability to below 30% in both assays. Additionally, the cytotoxicity of PVP-coated NPs was significantly lower than that of uncoated NPs of similar sizes.

Effect of CuO NPs on Intracellular Cu²⁺ ions

The AAS analysis demonstrated that the intracellular uptake of CuO NPs exhibited a dose-dependent pattern (Fig. 6). CuO-25 NP-treated groups exhibited significantly higher (P < 0.05) uptake per mg of cellular protein than CuO-48 NP-treated (P < 0.05) and CuO-P NP-treated groups, indicating a significant influence of size on the cellular uptake of NPs.

Effect of CuO NPs on cell membrane integrity

Damaged cell membranes promote cell death, which manifests as enhanced extracellular release of LDH enzyme. CuO NPs enhanced the LDH levels in the PC-12 cell culture supernatant (Fig. 7). At 1 μg/mL, the LDH release in the CuO-25 NP-treated and CuO-48 NP-treated groups was 40% (P < 0.05) and 31% (P < 0.05), respectively, at 96 h.

Treatment with 10 μg/mL CuO-25 NPs increased the LDH release rate to 43% (P < 0.05) and 71% (P < 0.05) at 24 and 96 h, respectively. The LDH release rate in cells treated with different concentrations of CuO-25 NPs at 24 and 96 h was as follows: 50 μg/mL: 60% (P < 0.05) and 91% (P < 0.01), respectively; 100 μg/mL: 75% (P < 0.01) and 91% (P < 0.01), respectively. Meanwhile, the LDH release rate at 24 and 96 h in cells treated with 50 μg/mL CuO-48 NPs was 46% (P < 0.05) and 78% (P < 0.01), respectively, while that in cells treated with 100 μg/mL CuO-48 NPs was 64% (P < 0.05) and 86% (P < 0.01), respectively. Comparative analysis revealed that the LDH release rate in the CuO-25 NP-treated group was significantly higher than that in the CuO-48 NP-treated group.

The LDH release rate in the CuO-P NP-treated group was significantly lower than that in the CuO-48 NP-treated group. At the maximum treatment concentration (100 μg/mL), the LDH release in the CuO-P NP-treated group was 53.55% (P < 0.05) and 77% (P < 0.01) at 24 and 96 h, respectively, which was significantly lower than that in the uncoated NP-treated groups. This indicated that PVP coating mitigated the cytotoxicity of CuO NPs.

Effect of CuO NPs on MMP

Treatment with 1 μg/mL CuO-25 and CuO-48 NPs decreased the MMP to 63% (P < 0.05) and 73% (P < 0.05), respectively, at 96 h (Fig. 8). Compared with that in the control group, the MMP decreased to 62% (P < 0.05) and 39% (P < 0.01) in the 10 μg/mL CuO-25 NP-treated group at 24 and 96 h, respectively. Meanwhile, the MMP decreased to 37% (P < 0.05) and 17% (P < 0.01) in the 50 μg/mL CuO-25 NP-treated group at 24 and 96 h, respectively. In the 100 μg/mL CuO-25 NP-treated group, the MMP decreased to 24% (P < 0.01) and 10% (P < 0.01) at 24 and 96 h, respectively. The MMP decreased to 54% (P < 0.05), 25% (P < 0.05), and 15% (P < 0.01) in the groups treated with 10, 50, and 100 μg/mL CuO48 NPs, respectively, at 96 h. The MMP decline was mitigated in the CuO-P NP-treated groups. In particular, the MMP decreased to 74% (P < 0.05), 62% (P < 0.05), and 58% (P < 0.05) in the groups treated with 10, 50, and 100 μg/mL CuO-P NPs, respectively, which was significantly lower than that in the groups treated with uncoated NPs.

Effect of CuO NPs on intracellular ROS and RNS production

The effects of 1 μg/mL CuO NP treatment for 72 h were evident in the cell viability assays. However, the ROS levels increased by 1.72-fold (P < 0.05) at 48 h only in the CuO-25 NP-treated group and remained steady till 96 h (Fig. 9). Treatment with 1 μg/mL CuO-48 NPs increased the ROS levels by 1.43-fold and 1.42-fold at 72 and 96 h, respectively. In contrast, CuO-P NPs did not increase the ROS levels.

At 10 μg/mL, CuO-25 NPs increased the ROS levels by 2.04-fold (P < 0.05) and 2.54-fold (P < 0.05) relative to the control at 24 and 96 h, respectively. Meanwhile, treatment with 50 and 100 μg/mL CuO-25 NPs increased the ROS levels by 3.32-fold (P < 0.01) and 4.07-fold (P < 0.01), respectively, at 96 h. The ROS levels increased by 2.44-fold (P < 0.01), 2.91-fold (P < 0.01), and 3.54-fold (P < 0.01) upon treatment with 10, 50, and 100 μg/mL CuO-48 NPs for 96 h, respectively, while those increased by 1.64-fold (P < 0.01), 2.35-fold (P < 0.01), and 3.02-fold (P < 0.01) upon treatment with 10, 50, and 100 μg/mL CuO-P NPs for 96 h, respectively.

Treatment with 1 μg/mL CuO-25 NPs and CuO-48 NPs increased the intracellular RNS levels at 48 and 72 h, respectively. However, treatment with 1 μg/mL CuO-P NPs did not increase the RNS levels (Fig. 10). The RNS levels increased by 1.76-fold and 2.01-fold at 24 and 96 h, respectively, upon treatment with 10 μg/mL CuO-25 NPs. Treatment with 50 and 100 μg/mL CuO-25 NPs increased the RNS levels by 2.43-fold and 2.88-fold, respectively, at 96 h. The RNS levels increased by 1.7-fold (P < 0.05), 2.17-fold (P < 0.01), and 2.5-fold (P < 0.01) after treatment with 10, 50, and 100 µg/mL CuO-48 NPs, respectively, for 96 h. Treatment with 10, 50, and 100 µg/mL CuO-P NPs for 96 h increased the RNS levels by 1.46-fold (P < 0.05), 1.58-fold (P < 0.05), and 2.14-fold (P < 0.01), respectively. Thus, at concentrations of ≥ 1 μg/mL, CuO-25 NPs upregulated the ROS and RNS levels when compared with CuO-48 and CuO-P NPs.

Effect of CuO NPs on Il6 and Tnf levels

Treatment with 1, 10, 50 and 100 μg/mL for 24 h CuO-25 NPs upregulated the Il6 levels by 3.38-fold (P < 0.05), 6.38-fold (P < 0.05), 11.66-fold (P < 0.01), and 14.41-fold (P < 0.01), respectively (Fig. 11a). Meanwhile, the Il6 levels were upregulated by 2.52-fold (P < 0.05), 5.04-fold (P < 0.05), 10.09-fold (P < 0.01), and 12.57-fold (P < 0.01) upon treatment with 1, 10, 50, and 100 μg/mL for 24 h. The exposure of CuO-P NPs did not affect the Il6 levels at 1 μg/mL. However, the Il6 levels were upregulated by 3.85-fold (P < 0.05), 7.80-fold (P < 0.01), and 10.14-fold (P < 0.01) upon treatment with 10, 50, and 100 μg/mL CuO-P NPs, respectively.

Treatment with 1 μg/mL CuO NPs did not affect the Tnf levels (Fig. 11b). The Tnf levels increased by 2.32-fold (P < 0.05), 3.60-fold (P < 0.05), and 4.47-fold (P < 0.001) upon treatment with 10, 50, and 100 μg/mL CuO-25 NPs, respectively. Meanwhile, treatment with 10, 50, and 100 μg/mL CuO-48 NPs upregulated the Tnf levels by 1.89-fold (P < 0.05), 2.91-fold (P < 0.05), and 3.80-fold (P < 0.05), respectively. CuO-P NPs did not affect the Tnf levels at 10 μg/mL. However, the Tnf levels increased by 2.43-fold (P < 0.05) and 3.29-fold (P < 0.05) upon treatment with 50 and 100 μg/mL CuO-P NPs, respectively.

Effect of CuO NPs on cellular apoptosis

Compared with negative control, treatment with 0, 50, and 100 μg/mL CuO-25 NPs for 24 h significantly upregulated Casp3 activity by 2.1-fold (P < 0.05), 2.56-fold (P < 0.001), and 2.93-fold (P < 0.001), respectively (Fig. 11c). Meanwhile, treatment with 10, 50, and 100 μg/mL CuO-25 NPs for 24 h increased Casp3 activity by 1.82-fold (P < 0.05), 2.32-fold (P < 0.05), and 2.68-fold (P < 0.001), respectively, which was significantly lower than that in the CuO-25 NP-treated group.

At 10 μg/mL, CuO-P NPs did not significantly affect Casp3 activity when compared with CuO-48 NPs. However, treatment with 50 and 100 μg/mL CuO-P NPs upregulated the Casp3 activity by 1.86-fold (P < 0.05) and 2.4-fold (P < 0.05), respectively, which was significantly lower than that in the CuO-48 NP-treated group.

Effect of CuO NPs on AChE activity

CuO NPs concentration-dependently inhibited AChE activity (Fig. 11d). Treatment with 10, 50, and 100 μg/mL CuO-25 NPs significantly inhibited AChE activity to 63% (P < 0.05), 45% (P < 0.05), and 24% (P < 0.01), respectively. Meanwhile, treatment with 10, 50, and 100 μg/mL CuO-48 NPs suppressed AChE activity to 71% (P < 0.05), 57% (P < 0.05), and 35% (P < 0.05), respectively. CuO-P NPs did not inhibit AChE activity at 10 μg/mL. However, treatment with 50 and 100 μg/mL CuO-P NPs inhibited AChE activity to 73% (P < 0.05) and 59% (P < 0.05), respectively.

Effect of CuO NPs on dopaminergic gene expression

Treatment with CuO-25, CuO-48, and CuO-P NPs for 24 h significantly upregulated the expression levels of Mao-A gene by 2.31-fold (P < 0.05), 2.21-fold, and 1.65-fold (P < 0.05), respectively (Fig. 12a). Additionally, treatment with CuO-25, CuO-48, and CuO-P NPs upregulated the expression of Th gene by 1.72-fold, 1.64-fold, and 1.66-fold, respectively. However, the Th gene expression levels were not significantly different between the CuO-25 NP-treated, CuO-48 NP-treated, and CuO-P NP-treated groups.

Next, three genes (Snca, Prkn, and Gpr37) associated with the etiology of NDs were analyzed. Treatment with CuO-25, CuO-48, and CuO-P NPs significantly upregulated α-synuclein expression by 3.53-fold (P < 0.05), 2.96-fold (P < 0.05), and twofold (P < 0.05), respectively (Fig. 12b). The Snca levels in the CuO-25 NP-treated group were significantly higher than those in the CuO-48 NP-treated group. However, unlike the Mao-A gene, a significant difference was observed between CuO-25 and CuO-48 NPs, with smaller NPs inducing significantly more up-regulation in gene expression compared to bigger counterparts.

Effect of released Cu²⁺ ions on cellular toxicity

The behavior of CuO NPs in the cell culture media was influenced by their size, exposure time, and surface coating. The disintegration of these NPs varied depending on these factors. CuO-25 NPs were associated with enhanced release of Cu²⁺ ions when compared with CuO-48 NPs (Fig. S4). Furthermore, CuO-P NPs exhibited the least Cu²⁺ ion release.

The released Cu²⁺ ions exerted neurotoxic effects at 10 μg/mL. At 96 h, cell viability decreased to 53% (P < 0.05) and 63% (P < 0.05) in the cells treated with culture media from the CuO-25 NP-treated and CuO-48 NP-treated groups, respectively (Fig. S5). Additionally, exposure to culture media from the CuO-25 NP-treated and CuO-48 NP-treated groups containing Cu²⁺ ions increased the ROS levels by 1.7-fold (P < 0.05) and 1.5-fold (P < 0.05), respectively (Fig. S6). The Il6 levels in the cells treated with culture media from the CuO-25 NP-treated and CuO-48 NP-treated groups increased by 1.85-fold (P < 0.05) and 1.65-fold (P < 0.05), respectively (Fig. S7). The Tnf levels in the cells treated with culture media from the CuO-25 NP-treated and CuO-48 NP-treated groups were upregulated by 3.1-fold (P < 0.05) and 2.42-fold (P < 0.05), respectively (Fig. S8). Furthermore, Casp3 activity was upregulated by 1.65-fold (P < 0.05) and 1.46-fold (P < 0.05) in the cells treated with culture media from the CuO-25 NP-treated and CuO-48 NP-treated groups, respectively (Fig. S9). The AChE activity decreased to 76% (P < 0.05) in the cells treated with culture media from the CuO-25 NP-treated group but was not affected in the cells treated with culture media from the CuO-48 NP-treated group (Fig. S10). Cell culture media containing the Cu²⁺ ions released from CuO-P NPs did not exert cytotoxic effects.

CuO NPs have various industrial and medicinal applications owing to their distinct physicochemical features. However, the adverse effects of CuO NPs on human health and the environment, especially their capacity to induce oxidative stress and damage biological components, have raised safety concerns^23,24. The accumulation level of CuO NPs in the brain tissue is unclear. Particles with a large size are generally considered safe as they cannot enter the brain owing to the BBB. However, efforts are ongoing to determine the maximal size of NPs that can traverse the BBB.

Similar to other metal NPs, CuO NPs are reported to exert neurotoxic effects. CuO NPs induce oxidative stress, which can damage macromolecules, such as lipids, proteins, and DNA^25,26, adversely affecting metabolic activity, cell viability, and neuronal structure. However, conflicting findings have been reported on the cytotoxicity of CuO NPs. One study reported that CuO NPs (53 nm) do not decrease the viability of MCF-7 cells (breast carcinoma cells) below 50% even after treatment at a high concentration of 1600 μg/mL for 24 h²⁷. Similar outcomes were observed in the N2A mouse neuroblastoma cell line exposed to CuO (102 ± 34 nm). The viability of neuroblastoma cells did not decrease even at 400 mg/L after 24 h of exposure²⁸. This can be explained by the agglomeration of NPs in cells. Previous studies did not perform DLS or zeta potential analysis. The sonication of NPs before treatment is essential and can significantly affect the outcomes²⁹ However, one study reported decreased viability and Cu accumulation in C6 glioma cells³⁰. The findings of this study are consistent with those reported in the previous study. CuO NPs decreased cell viability in a time-dependent, concentration-dependent, size-dependent, and coating-dependent manner. Treatment with 1 μg/mL CuO-25 NPs for 72 h decreased cell viability by 25–30%, exerting the maximum cytotoxicity. Meanwhile, CuO-48 NPs exerted the second highest cytotoxic effects. The MTT, NRU, and LDH assay results revealed that PVP coating mitigated the cytotoxicity of NPs.

Neuronal function is dependent on MMP. The primary source of energy for the neurons is the mitochondria in which energy is generated through oxidative phosphorylation³¹. MMP is critical for neuronal function and survival. Neurons require a steady supply of adenosine triphosphate (ATP) to maintain membrane potential and release neurotransmitters³². Mitochondrial dysfunction can lead to impaired ATP synthesis, oxidative stress, and cell death, as well as decreased MMP³³. In this study, CuO NPs differentially impaired MMP. The MMP decreased to 63% and 75% upon treatment with 1 μg/mL CuO-25 NPs and CuO-48 NPs for 96 h, respectively. However, the MMP in the CuO-P NP-treated group was significantly higher than that in the uncoated NP-treated group.

MMP decline is a major inducer of oxidative stress, disrupting the electron transport chain and increasing the formation of ROS, such as superoxide anions and hydroxyl radicals³⁴. ROS can damage cellular macromolecules, such as lipids, proteins, and DNA, and activate various signaling pathways that result in cell death³⁵. Additionally, nitric oxide (NO) is the major RNS produced by NO synthases. NO can combine with other molecules to form other RNS, such as peroxynitrite (ONOO-), nitrogen dioxide (NO₂), and nitrous oxide (N₂O₃). RNS is involved in several physiological and pathological processes in brain cells, including neurotransmission, synaptic plasticity, and inflammation³⁶. Excess RNS production can induce oxidative and nitrosative stress, which can promote cellular damage and contribute to the development of various neurological disorders³⁷. This study demonstrated the correlation between MMP decline and elevated ROS in the effect of NPs, which was concentration-dependent and time-dependent. CuO NPs significantly upregulated the ROS and RNS levels in PC-12 cells. The ROS and RNS levels were the highest in the CuO-25 NP-treated group and the lowest in the CuO-P NP-treated group. These findings were consistent with those of previous studies, which reported that CuO NPs upregulated a specific pro-apoptotic gene (Bax) and downregulated an anti-apoptotic gene (Bcl2) in the mouse hippocampus HT-22 cell line. Additionally, CuO NPs downregulated the activity of various detoxification enzymes (glutathione S-transferase and superoxide dismutase)³⁸. An in vivo study reported increased oxidative stress in the murine brain post-intra-nasal exposure to 23.5 nm CuO NPs³⁹. The increased oxidative stress in the brain of rats orally administered with CuO NPs was due to the downregulation of cytochrome P-450 enzymes⁴⁰.

Pro-inflammatory cytokines, such as TNF-α and IL-6 have critical roles in immune responses. Additionally, pro-inflammatory cytokines are involved in various physiological and pathological processes in the brain, including learning and memory⁴¹. Under physiological conditions, TNF-α is produced in small quantities to regulate neurogenesis, synaptic plasticity, and neuroprotective mechanisms⁴². TNF-α and IL-6 activate various signaling pathways, including the NF-κB and MAPK pathways, promoting the production of ROS, the activation of adhesion molecules, and the migration of immune cells to the brain⁴³. Furthermore, TNF-α and IL-6 may regulate synaptic plasticity, cognition, and neurotransmitter systems, such as glutamate and gamma-aminobutyric acid⁴¹. The dysregulation of TNF-α and IL-6 can lead to the development of various neurological disorders, such as Alzheimer’s and Parkinson’s disease⁴⁴. RNS and ROS synergistically promote the release of pro-inflammatory mediators. Consistently, this study demonstrated that treatment with 10 μg/mL CuO NPs significantly upregulated the levels of the pro-inflammatory cytokines Il6 and Tnf in PC-12 cells. The upregulation levels of Tnf and Il6 were dependent on the size and coating of the NPs. In particular, the levels of Tnf and Il6 were the highest in the CuO-25 NP-treated group, followed by the CuO-48 NP-treated and CuO-P NP-treated groups. These results were supported by the findings of a previous study, which reported that prostaglandin E₂, Tnf, and Il1b were upregulated in rat brain microvessel endothelial cells exposed to CuO NPs (40 and 60 nm) for 8 h⁴⁵.

The activation of caspase-3, which is involved in programmed cell death or apoptosis, is one of the downstream effects of the upregulation of pro-inflammatory cytokines. Under physiological conditions, caspase-3 exists as an inactive proenzyme. However, caspase-3 is cleaved and activated in response to specific signals⁴⁶. Caspase-3 activation leads to DNA fragmentation and the breakdown of cellular components. This mechanism is tightly regulated to prevent excessive cell death, which can cause tissue damage and disease. CuO NPs upregulated caspase-3 activity in SH-SY5Y, H4, and PC-12 cells¹². The findings of this study are consistent with these findings. Casp3 activity was the highest in the CuO-25 NP-treated group, followed by the CuO-48 NP-treated group (at NP concentrations of ≥ 10 μg/mL). In contrast, CuO-P NPs did not upregulate Casp3 activity at 10 μg/mL but upregulated its activity at 50 and 100 μg/mL.

CuO NPs concentration-dependently inhibited AChE activity, which may be the potential mechanism underlying their neurotoxicity. Treatment with CuO-25 NPs at concentrations ≥ 10 μg/mL inhibited AChE activity. In contrast, CuO-48 and CuO-P NPs inhibited AChE activity to a lesser degree when compared with CuO-25 NPs, suggesting a significant size-dependent effect. This difference in AChE inhibitory activities can be attributed to differential NP size and surface characteristics, which can affect their interaction with the enzyme⁴⁷. AChE inhibition is a biomarker of neurotoxicity as it prevents the breakdown of acetylcholine, a critical neurotransmitter for synaptic communication⁴⁸. The prolonged presence of acetylcholine in the synaptic cleft can overstimulate neuronal circuits, contributing to nicotinic and muscarinic toxicity⁴⁹. CuO-P NPs did not affect AChE activity at 10 μg/mL but significantly inhibited AChE activity at 50 and 100 μg/mL. This indicates that PVP coating may partially prevent AChE inhibition.

CuO NP exposure upregulated the expression of genes involved in dopamine metabolism and NDs. In particular, CuO-25 and CuO-48 NPs upregulated the expression of MaoA gene. The upregulation of MaoA gene in the CuO-P NP-treated group was lower than that in the CuO-25 NP-treated and CuO-48 NP-treated groups. This suggests that surface coating moderately mitigates MaoA gene upregulation. The upregulation of MaoA gene alters dopamine metabolism, which may have implications for neurotransmitter modulation and potentially lead to neurotoxicity⁵⁰. This is consistent with the findings of previous studies, which reported that metal ions, such as Cu, alter dopamine homeostasis and contribute to neurotoxicity^7,51.

Furthermore, CuO NP exposure upregulated α-synuclein expression. α-synuclein is associated with the etiology of neurodegenerative diseases, including Parkinson’s disease⁵². CuO-25 and CuO-48 NPs differentially upregulated α-synuclein expression. In particular, the upregulation of α-synuclein induced by CuO-25 NPs was higher than that induced by CuO-48 NPs, indicating increased neurotoxicity of particles with small sizes. Thus, CuO NPs may contribute to the development of neurodegenerative diseases through the upregulation of α-synuclein. This finding is consistent with the emerging evidence suggesting a role for metal NPs, including Cu, in the aggregation and toxicity of α-synuclein. However, further studies are needed to elucidate the underlying mechanisms and implications for neurodegenerative processes^53,54.

Excessive Cu²⁺ ion levels in the brain can lead to NDs, including Wilson’s disease. Cu²⁺ ions released from NPs can exert toxic effects by inducing oxidative stress, disrupting the neurotransmitter systems, and promoting protein aggregation. CuO NPs release Cu²⁺ ions when they come in contact with biological fluids or cells⁵⁵. The free Cu²⁺ ions can interact with various biological components, exerting cytotoxic effects and inducing oxidative stress⁵⁶. One study reported that the release of Cu²⁺ ions from NPs was higher than that from micrometer-sized particles. However, the cytotoxic effects of Cu²⁺ ions released from NPs were lower than those of NPs⁵⁷. In this study, AAS analysis revealed that the release of Cu²⁺ ions in the cell culture media was upregulated in the CuO NP-treated group. The released Cu²⁺ ions mediated the neurotoxic effects of CuO NPs. The exposure of PC-12 cells to culture media (containing released Cu²⁺ ions) of CuO-25 NP-treated and CuO-48 NP-treated groups decreased cell viability and increased ROS, Il6, and Tnf levels. Additionally, the Cu²⁺ ions in the culture media upregulated the activity of caspase-3, a marker of apoptosis, confirming the negative effects of the ions on neural PC-12 cells. Thus, excess Cu²⁺ ions can lead to neurotoxicity and other NDs. However, further studies are needed to examine the effects of excess or deficient Cu on ND etiology^58,59. Cu²⁺ ions released from CuO-25 NPs significantly inhibited AChE activity, indicating potential cholinergic signaling disruption, which is associated with various neurodegenerative disorders (NDs).

The mechanisms underlying CuO NP-induced neurotoxicity are complex and involve various pathological processes, including oxidative stress, inflammation, mitochondrial dysfunction, and protein misfolding^60,61. Oxidative stress is one of the key mechanisms through which CuO NPs exert neurotoxic effects. CuO NPs can promote protein oxidation, lipid peroxidation, and DNA damage through the Fenton and Haber–Weiss reactions⁶². ROS can destabilize MMP and trigger cytochrome-c release, leading to apoptosis induction. In this study, CuO NP-induced neurotoxicity was dependent on particle concentration, physical diameter, and surface coating. However, further studies are needed to elucidate the mechanism underlying CuO NP-induced neurotoxicity. These mechanisms have been implicated in the pathophysiology of a number of neurodegenerative illnesses, including as Parkinson’s, Alzheimer’s, and prion disorders, which are all marked by the build-up of aggregated or misfolded proteins⁶³.

It is important to acknowledge the limitations of the 2D cell culture model used in this study. While PC-12 cells provide valuable insights into nanoparticle-induced cytotoxicity and neurotoxic mechanisms, 2D systems lack the three-dimensional architecture, extracellular matrix interactions, and physiological gradients present in-vivo. These factors may influence the extent of CuO nanoparticle uptake, mitochondrial stress, and downstream neurotoxic outcomes observed in our study. For example, the dose-dependent decline in mitochondrial membrane potential and increase in intracellular Cu accumulation reported here may differ in magnitude or kinetics in more physiologically relevant systems. Future work using 3D neural cultures or in vivo models will be essential to validate and extend these findings.

CuO NP-induced neurotoxic effects can be attributed to several factors, including concentration, particle size, surface coating, and the release of Cu²⁺ ions. These properties influence the cellular uptake of NPs and their interaction with cellular components as well as their propensity to induce oxidative stress. Although CuO NPs have shown neurotoxic effects in both in- vitro and in- vivo models, it is crucial to remember that the doses used in in- vitro studies frequently surpass physiologically relevant levels, especially given the limited ability of nanoparticles to translocate across the BBB after systemic exposure. According to the literature, due to the barrier’s limiting properties and intricate biodistribution dynamics, there is usually little real accumulation of nanoparticles in brain tissue after crossing the BBB. As a result, care must be taken when extrapolating concentration–response results from in- vitro experiments to in- vivo exposure situations. We acknowledge this limitation and emphasize that our findings represent a first step in understanding nanoparticle -neuron interactions, primarily providing mechanistic insights under carefully controlled experimental conditions.