Carbonaceous materials derived from corn cob waste via pyrolysis, hydrothermal carbonization and open burning: contrasting biocompatibility and Cu²⁺ neurotoxicity modulation in a synaptosome model

Abstract

Here, three different types of carbonaceous nanoparticles/materials were obtained from corn cob agricultural waste using different “green” approaches without surface functionalization. Biochars synthesized from corn cobs by two-step pyrolysis at 600 °C (PB), carbon dots synthesized using a hydrothermal approach at 180 °C (CD) and smoke particulate matter (SP) prepared by open burning were comparatively analyzed for biocompatibility and Cu²⁺ adsorption capacity using a presynaptic rat cortex nerve terminal model (synaptosomes). The PB did not change the extrasynaptosomal levels of inhibitory neurotransmitter [³H]GABA and excitatory neurotransmitter L-[³H]glutamate within the concentration range of 0.1–1.0 mg mL⁻¹, while the CD and SP increased the extrasynaptosomal levels of these neurotransmitters starting from the concentrations of 0.5 and 0.1 mg mL⁻¹, respectively. In a fluorimetric study, the PB did not change the mitochondrial membrane potential of nerve terminals, while both the CD and SP significantly depolarized the mitochondrial membrane. Using an animal model of acute heavy metal-induced neurotoxicity, different capability of carbonaceous materials to influence Cu²⁺-induced neurotoxicity was found. In particular, the PB significantly mitigated Cu²⁺-induced damaging effects on the extrasynaptosomal neurotransmitter levels, while the CD and SP further increased Cu²⁺-induced neurotoxicity in a synergistic manner. In fluorimetric measurements of the plasma membrane and mitochondrial membrane potential, the PB mitigated Cu²⁺-induced membrane depolarization, while the CD and SP further depolarized the membranes of nerve terminals in a synergistic manner. Moreover, the PB and CD/SP showed different effects on ROS generation in nerve terminals. Therefore, three carbonaceous nanoparticles/materials obtained from corn cob waste using different “green” methods demonstrated different biocompatibility profiles and opposite modulatory effects on Cu²⁺-induced neurotoxicity in the nerve terminal model.

Here, three different types of carbonaceous nanoparticles/materials were obtained from corn cob agricultural waste using different “green” approaches without surface functionalization.

1. Introduction

The increasing accumulation of agricultural waste and the growing demand for sustainable nanomaterials have intensified the interest in converting biomass residues into carbonaceous materials. Among agricultural by-products, corn cobs represent an abundant, renewable, and low-cost waste that is often utilized or disposed of by open burning, contributing to environmental pollution and particulate matter emissions.¹ With global corn production exceeding 1.31 billion tons per year, corn cobs are estimated to account for about 15–20% of the dry mass relative to harvested grain. This massive and continuous waste stream highlights the urgency of developing conversion pathways, leading to valuable products.^1–4

Corn cobs are a lignocellulosic biomass composed mainly of cellulose, hemicellulose (predominantly xylan), and lignin. Increasing attention has been paid to the transformation of corn cob waste into cellulose nanomaterials with potential biomedical applications.^5,6 One of the most actively explored routes is the extraction of cellulose and its conversion into nanocellulose, including cellulose nanocrystals and cellulose nanofibers. Corn cobs can be subjected to delignification and bleaching, followed by acid hydrolysis or mechanical fibrillation, to yield nanoscale cellulose with high crystallinity, large specific surface area, and satisfactory mechanical properties. Corn cob-derived nanocellulose has demonstrated good biocompatibility, biodegradability, and low cytotoxicity, which are key requirements for biomedical use.^5,6 These materials are increasingly investigated for the fabrication of hydrogels and membranes, as well as wound dressing and antibacterial applications.^7,8 In addition, xylan extracted from corn cobs has been used as a natural reducing and stabilizing agent for the green synthesis of metal nanoparticles, such as silver nanoparticles.⁹

The conversion of biomass into carbonaceous nano- and micro-sized particles or nanoporous materials is another route of its utilization. Such carbonaceous materials can be produced through diverse thermochemical processes, including pyrolysis and hydrothermal carbonization. Uncontrolled combustion of agricultural waste is one more process, resulting in the formation of carbonaceous species. These processes yield materials with markedly different physicochemical properties, ranging from porous biochars to fluorescent carbon dots and carbonaceous smoke particulate matter, respectively.^10–13 Such differences can strongly influence the environmental behavior of such materials, their biological interactions, and adsorption performance toward toxic contaminants, particularly heavy metals, and prospects for application in environmental remediation.^12–14

The biochar produced via controlled pyrolysis is a micro- to meso-porous carbonaceous material widely studied for soil amendment, pollutant immobilization, and water purification.^15,16 Its high surface area, aromatic carbon structure, and surface functional groups enable effective adsorption of heavy metals such as lead, cadmium, and copper.^17–19 In our recent study, we have confirmed the biocompatibility and high heavy metal adsorption capacity of the biochar from corn cobs synthesized at 800 °C.²⁰ It was demonstrated that corn cob-derived activated carbon could act as an efficient lead adsorbent, its performance was associated with the porous texture and presence of functional groups, e.g., carboxyl, hydroxyl, and amino groups.²¹ Despite its perceived environmental safety, biochar particles may still interact with biological systems, and their potential neurobiological effects remain poorly understood.

Hydrothermal carbon dots are discrete particles, typically below 10 nm in diameter, enriched with oxygen- and nitrogen-containing surface functional groups that confer water dispersibility, photoluminescence, and potential biocompatibility.²² Due to these features, biomass-derived carbon dots have attracted attention for applications in bioimaging, sensing, and environmental remediation. According to our data, carbon dots from β-alanine and thiourea/citric acid have demonstrated neurotropic action.^23,24 However, their interaction with neuronal cells remains insufficiently characterized, particularly in comparison with larger carbonaceous particles originating from the same precursor material.

Carbonaceous smoke particulate matter is generated during biomass combustion. Nerve cell exposure to biomass-derived particulate matter has been associated with oxidative stress, inflammation, and neurotoxicity.^25,26 In our previous study, it has been clearly demonstrated that carbonaceous smoke particulate matter from different types of wood and plastics impaired neurotransmitter transport and membrane mitochondrial potential in a nerve terminal model, and hence, possessed neurotoxic signs.²⁷

Among biological models, isolated nerve terminals (synaptosomes) represent a sensitive and well-established ex vivo model, and its use allows reliable and quick assessment of acute neurotoxicity, oxidative damage, and disruptions in neurotransmitter transport.²⁸ Evaluating carbonaceous materials in this model provides mechanistic insights into their potential neurological risks while avoiding different distorting factors present in vivo studies. Nevertheless, direct comparative studies examining the biocompatibility of carbonaceous combustion-derived particulate matter alongside engineered carbon nanomaterials, such as pyrolysis biochars and carbon dots produced from the same biomass source are scarce.

In this work, we performed comparative investigation of the different carbonaceous nano- and micro-sized materials derived from corn cob waste, including pyrolysis biochars (PB), hydrothermal carbon dots (CD), and smoke particulate matter (SP). The study focused on their physicochemical characteristics, biocompatibility in a nerve terminal model, and heavy metal adsorption capacity. By directly comparing materials produced from the same precursor via distinct thermochemical pathways, this study aims to elucidate how production method and particle surface properties influence both functionality and neurobiological safety.

Taking into account the abovementioned facts, the aims of the present study are as follows: (1) to synthesize three principally different types of “green” carbonaceous micro- and nano-particles/materials from corn cob waste without special surface functionalization, i.e. PB obtained at 600 °C, CD synthesized using a hydrothermal approach at 180 °C and water-suspended SP collected during all firing phases; (2) to compare the biocompatibility of PB, CD and SP in biological model using presynaptic rat cortex synaptosomes and analyzing the extrasynaptosomal levels of key inhibitory and excitatory neurotransmitters in the central nervous system, [³H]GABA and L-[³H]glutamate, respectively; and (3) to examine comparatively the Cu²⁺ adsorption capacity of these nanoparticles/materials in the nerve terminal model.

Previously, we have developed a method for the preparation of a series of pyrolysis biochars and found that an increase in pyrolysis temperature along with high-temperature treatment with NaOH resulted in a significant increase in the specific surface area, but a drastic decrease in the content of functional groups on the surface of biochars. Though the biochar, prepared at 800 °C by a two-step procedure, had the best performance in the sorption of Cu²⁺ ions,²⁰ its preparation method was significantly more energy- and resource-consuming than the simpler analogue (prepared at 600 °C by a one-step procedure without any need for NaOH wash), and its high cost can be a serious obstacle for application of this sorbent. Thus, in this study we used the biochar, prepared at 600 °C without treatment with NaOH.

2. Methods and materials

2.1. Synthesis of the PB from corn cob waste

The biochar was synthesized at 600 °C, as described in our recent paper.²⁰

2.2. Synthesis of the CD from corn cob waste

The CD were synthesized by a hydrothermal method according to the procedure reported previously,²⁹ with some modifications described below. First, 50 mL of distilled water was added to 0.5 g of corn cob powder and stirred for 70 min. The homogeneous suspension was placed in an 80 mL Teflon beaker of an autoclave, and hydrothermal treatment was carried out for 12 hours at a temperature of 180 °C. Then the content of the Teflon beaker was cooled down to room temperature, transferred to centrifuge tubes, and centrifuged twice for 30 min at 5500 rpm. After decantation, the resulting solution was filtered through a paper filter. Subsequent centrifugation under the same conditions did not result in the formation of a precipitate. The solution of CD was brown-yellow in color and exhibited yellow-green luminescence under UV light.

2.3. Methods for the characterization of the synthesized materials PB and CD

The TEM images of the synthesized materials were obtained using a TEM 125K (Selmi) instrument at an accelerating voltage of 100 kV. The samples were suspended in water, and the suspension was dropped onto a Cu grid (300 mesh) covered by an amorphous carbon film and dried on air.

Examination by SEM was performed using an FEI Inspect instrument. For the examination of PB, a suspension of the material was dropped onto a conducting carbon film without additional treatment and dried in air. The measurements were performed at 30 kV. For examination of the CD, the colloid of the CD was deposited on the surface of a silicon single crystal and dried in air at ambient temperature. The measurements were performed at 15 kV.

The IR spectra of materials were measured using a PerkinElmer Spectrum One instrument, for the measurements dry samples were pressed in the pellets with KBr. The optical spectra of the colloidal solution of the CD were recorded using a Unico SpectroQuest 4802 UV/vis spectrometer.

2.4. Synthesis of water-suspended SP from corn cob waste

SP was obtained under laboratory conditions by collecting the entire flaming, mixed (flaming and smoldering) and smoldering combustion phase. The obtained preparations were cleaned according to previous work.^30,31 In detail, smoke emission from the combusting dry corn cobs was collected using a self-designed vacuum installation, bubbled via water (50 mL), and then filtered through a glass microfiber filter with a pore diameter of 1.0 µm. The emission of PM was monitored according to previous work.^27,31,32 Then, SP was dried to measure the concentration of smoke particulate components. In SP, the concentration of dry components was ∼4.5 µg mL⁻¹.

Water-suspended smoke preparations obtained from different organic wastes using the same approach have been well-characterized in our previous studies using dynamic light scattering and IR spectroscopy.^27,31–33

2.5. Animal model

2.5.1. Ethics

Male Wistar rats aged three months were housed at 22–23 °C in a quiet, temperature-controlled vivarium at the Palladin Institute of Biochemistry. The animals had free access to standard dry chow and water. All animal procedures complied with the European Community Directive 2010/63/EU, the scientific requirements and research protocols, the principles of the Declaration of Helsinki, the ARRIVE guidelines for animal research reporting,^34,35 and the relevant Ukrainian legislation. Experimental protocols were approved by the Animal Care and Use Committee of the Palladin Institute of Biochemistry (Protocol No. 1, dated 15 January 2025). A total of 24 animals were used in the study.

2.5.2. Presynaptic nerve terminals (synaptosomes) isolated from the rat cortex

Nerve terminals were isolated from the cerebral cortex of rat brains. All procedures were performed at 4 °C. The cortical tissue was dissected and homogenized in an ice-cold buffer containing 0.32 M sucrose, 5 mM HEPES–NaOH (pH 7.4), and 0.2 mM EDTA. One synaptosome preparation was obtained from each rat. Isolation was carried out using a modified Cotman method^36–38 based on differential centrifugation and Ficoll-400 density gradient separation. The standard saline solution used in synaptosome experiments consisted of 126 mM NaCl, 5 mM KCl, 2.0 mM MgCl₂, 1.0 mM NaH₂PO₄, 20 mM HEPES (pH 7.4), and 10 mM d-glucose. The protein concentration was determined according to the method of Larson et al.³⁹ using a Shimadzu UV-1900i spectrophotometer. Synaptosomes accounted for approximately 87% of particles in the preparations and were free of neuronal cell bodies and functionally intact glial fragments.^40,41

2.5.3. Extrasynaptosomal level of [³H] GABA

Synaptosomes (2 mg protein per mL) were preincubated at 37 °C for 10 min and subsequently preloaded with [³H]GABA (1 µCi mL⁻¹) in a standard saline solution at 37 °C for an additional 10 min. Aminooxyacetic acid (100 µM) was present in the incubation medium throughout all experiments. The preparations were then washed with ten volumes of ice-cold standard saline solution, and the pellets were resuspended to a final protein concentration of 1 mg mL⁻¹.

Synaptosomal aliquots were preincubated for 8 min, after which the PB, CD, SP, CuSO₄ and their combinations (preincubated for 30 min in water) were added, and the suspension was incubated at 37 °C during 0 and 15 min (the measurements were performed in the presence of the particles in the synaptosomal incubation media), followed by centrifugation at 10 000×g for 20 s at room temperature.⁴² Extracellular [³H]GABA levels were determined in 90 µL aliquots of the supernatant by liquid scintillation counting using Sigma-Fluor® High Performance LSC Cocktail and a Hidex 600SL liquid scintillation counter (Finland). Extracellular [³H]GABA was expressed as the percentage of the total [³H]GABA accumulated by the synaptosomes. The data were obtained in triplicate.

2.5.4. Extrasynaptosomal level of L-[³H]glutamate

Synaptosomes (2 mg protein per mL) were preincubated at 37 °C for 10 min and subsequently loaded with L-[³H]glutamate (1 µCi mL⁻¹) at 37 °C for an additional 10 min. The preparations were then washed with ten volumes of ice-cold standard saline solution and centrifuged at 10 000×g for 20 s at 4 °C. The resulting pellets were resuspended in standard saline to a final concentration of 1 mg protein per mL.

Extracellular L-[³H]glutamate levels were assessed in 125 µL synaptosomal aliquots (0.5 mg protein per mL). The samples were preincubated for 8 min to allow the restoration of ion gradients, after which the PB, CD, SP, CuSO₄ and their combinations were added, and the suspension was incubated at 37 °C for 0 and 15 min. The samples were then centrifuged at 10 000×g for 20 s at room temperature. Extracellular L-[³H]glutamate was quantified in 100 µL aliquots of the supernatant, while radioactivity associated with synaptosomal pellets, pre-treated with 100 µL of 10% SDS, was measured by liquid scintillation counting using Sigma-Fluor® High Performance LSC Cocktail and a Hidex 600SL liquid scintillation counter (Finland).⁴³ The data were obtained in triplicate.

2.5.5. Synaptosomal plasma membrane potential

Rhodamine 6G (0.5 µM), a fluorescent potentiometric dye, was used to monitor the plasma membrane potential of nerve terminals. Synaptosomal suspensions (0.2 mg protein per mL) were preincubated at 37 °C for 10 min. The resulting supernatants were then added to synaptosomal suspensions previously incubated with rhodamine 6G.

In control experiments, a standard saline solution was added to the synaptosomal incubation medium. Rhodamine 6G fluorescence was measured at an excitation wavelength of 528 nm and an emission wavelength of 551 nm using a Shimadzu RF-6000 spectrofluorimeter.

2.5.6. Synaptosomal mitochondrial membrane potential

Mitochondrial membrane potential in synaptosomes was assessed using JC-1, a cationic, membrane-permeable fluorescent dye (mitochondrial membrane potential assay kit), to evaluate the potential changes induced by the PB, CD, SP, CuSO₄, and their combinations. At low mitochondrial membrane potential, JC-1 predominantly exists in the monomeric form, exhibiting green fluorescence with an emission maximum at ∼530 nm, while at high membrane potential, the dye forms aggregates, producing red-orange fluorescence with an emission maximum at ∼590 nm. The dissipation of the mitochondrial membrane potential results in a shift from red (J-aggregate) to green (monomeric) fluorescence.

In the present study, JC-1 accumulation by synaptosomal mitochondria occurred in a membrane potential-dependent manner, as evidenced by green fluorescence of the monomeric form (λ_em ≈ 529 nm) and the formation of red fluorescent J-aggregates with a concentration-dependent red shift to λ_em ≈ 590 nm. Aliquots of synaptosomal suspensions (0.15 mg protein per mL) were incubated with JC-1 (5 µM) at 37 °C for 10 min in the dark under continuous stirring. Fluorescence spectra were recorded at an excitation wavelength of 485 nm and emission wavelengths ranging from 510 to 610 nm.

The PB, CD, SP, CuSO₄, and their mixtures (preincubated for 30 min in water) were subsequently added to the cuvette, and fluorescence was recorded after 30 min of incubation at 485 nm excitation and 590 nm emission (measurements were performed in the presence of biochars in the synaptosomal incubation medium). The changes in JC-1 fluorescence spectra induced by the PB, CD, SP, CuSO₄, and their mixtures were analyzed at 485 nm excitation with emission scanned from 510 to 610 nm using the Hitachi 650-10S and Shimadzu RF-6000 spectrofluorimeters.

For quantitative analysis, the ratio of fluorescence intensities at 590 and 530 nm was calculated. This ratiometric approach minimized the influence of variations in dye loading, mitochondrial content, potential nonspecific interactions of JC-1 with the PB, CD, SP and CuSO₄, as well as fluorescence quenching caused by these agents and their combinations.

2.5.7. Synaptosomal ROS generation

Intracellular reactive oxygen species (ROS) production in synaptosomes was assessed using the cell-permeable nonfluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCFDA, 5 µM). Following intracellular de-esterification and subsequent oxidation, H2-DCFDA is converted to the fluorescent product 2′,7′-dichlorofluorescein (DCF). Synaptosomal aliquots (0.2 mg protein per mL) were preincubated with H2-DCFDA at 37 °C for 3 min, after which the effects of the PB, CD, SP, CuSO₄, and their mixtures (preincubated for 30 min in water) were assessed.

DCF fluorescence was recorded at an excitation wavelength of 502 nm and an emission wavelength of 525 nm (slit width = 2 nm) using a fluorescence spectrofluorimeter (QuantaMaster™ 40, PTI Inc., Canada).²⁷

2.6. Statistical analysis

The data of the experiments are expressed as mean ± S.E.M. of n independent experiments. In order to test the normality in samples, the Shapiro–Wilk procedure was used. In order to verify the variance homogeneity for the groups (p > 0.05) before applying ANOVA, Levene's test was used. One-way ANOVA was applied, and the accepted significance was p < 0.05. All experimental data on the assessment of the effects of the PB, CD and SP on the Cu²⁺-induced neurotoxicity were analyzed by two-way ANOVA followed by Tukey's post hoc test, where the PB/CD/SP and copper were used as independent factors.

2.7. Materials

In animal experiments, HEPES, EGTA, EDTA, Ficoll 400, analytical-grade salts, and High Performance LSC Cocktail were purchased from Sigma (USA). L-[³H]glutamate and [³H] GABA were obtained from Revvity (Waltham, MA, USA).

3. Results

3.1. Physical and chemical characterization of the PB and CD from corn cobs waste

PB and CD materials were prepared by thermolysis of the corn cobs in different conditions, i.e. heating in an inert atmosphere at 600 °C (PB) and hydrothermal treatment at 180 °C (CD), respectively. According to EDX analysis, these samples mainly consisted of carbon, oxygen and nitrogen, and expectedly contained residues of bioelements (Na, Mg, P, etc.), present in corn cobs (Table 1). In these materials, oxygen mainly originates from the functional groups, such as carboxy, carbonyl, hydroxy (including phenolic), and epoxide, while the presence of nitrogen confirms the formation of nitrogen-containing functional groups or N-doping of the carbonaceous material, and it agrees with the reported data.⁴⁴ The carbon content in PB was significantly higher than that in CD (ac. 80% vs. 48%), while the oxygen content was higher in CD (ca. 36% vs. 14%). A similar tendency was found for the content of nitrogen – it is higher in the CD sample (4.9%) than in the PB sample (3.6%). As indicated above, such difference can be a sign of significantly higher content of oxygen- and nitrogen-containing functional groups in the CD sample, which is consistent with a significantly lower CD formation temperature. The increase in carbonization temperature leads to the thermal (pyrolytic) destruction of a larger quantity of functional groups.⁴⁵

Table 1. Elemental content in the PB and CD measured by EDX (in weight%).

	C	O	N	Na	Mg	P	S	Cl	K	Ca
PB	79.8	13.5	3.6	1.4	0.3	0.2	0.3	0.3	0.7	0.1
CDa	47.8	36.4	4.9	2.9	0.7	0.4	0.4	5.2	1.0	0.2

^aAverage of 5 measurements.

Other elements originate from the bioelements, which were present in the raw materials and formed non-volatile compounds upon thermal decomposition. Notably, the increased content of Cl, S, and P in CD (which presumably exists in the considered materials as chloride, sulfate and phosphate anions, respectively) goes in line with the increased content of the elements, which presumably form the cations Na, K, Mg and Ca; such tendency is consistent with the electrostatic balance demand.

The morphology of the carbonaceous material PB was studied by TEM and SEM. The sample consisted of particles of irregular shape with a size of ca. 20 nm (Fig. 1), which formed aggregates of size up to 200–300 µm (which can be distinguished by SEM; Fig. 2). One of the aggregates of ca. 1 µm is shown in Fig. 1. Such aggregates are arranged in such a way that the voids with a size of 10–30 µm can be observed. While some of these voids can reflect the cellular structure of the initial corn material (such as the one shown in Fig. 2b), others apparently formed due to the spontaneous aggregation of particles upon carbonization (Fig. 2a and c).

Fig. 1. TEM image of the carbonaceous nanomaterial PB.

Fig. 2. SEM images of the carbonaceous nanomaterial PB under different magnifications of (a) 100 µm, (b) 20 µm and (c) 10 µm.

For the study by SEM, a colloidal solution of CD was deposited in microdroplets on the surface of a silicon single crystal. The micrographs were measured in two detection modes – the secondary electron (SE; Fig. 3a, c and d) and back-scattered electron mode (BSE; Fig. 3b). In the SE mode, the morphology of the surface of the material is visible, while in the BSE mode, the phase contrast is better manifested, where the contrast brightness of the objects is proportional to the atomic mass of the elements.

Fig. 3. SEM images of the CD deposited from a colloidal solution under different magnifications in the (a) SE mode, (b) BSE mode, and (c) and (d) SE mode.

Two types of materials can be distinguished on the SEM images of the CD: round “plates” consisting of light elements (dark spots in the BSE mode; Fig. 3b) and needle-like aggregates containing heavier materials (bright spots in the BSE mode). The former can be assigned to the carbonaceous materials, while the latter probably represent crystals of inorganic salts, which were “washed out” from the corn raw materials (such as NaCl or MgCl₂; the presence of Na, Mg and Cl was confirmed by EDX). We failed to achieve complete removal of these salts, and it can be supposed that their binding with the carbonaceous materials is facilitated by sorption (presumably by ion-exchange mechanism). Since the impurities are mainly inorganic salts of bio-elements (which were present in the starting corn cobs), they are not toxic and their effect on nerve terminals is negligibly small.

All round “plates” of light elements observed in the SEM images have different sizes; the largest have a diameter of some dozens of micrometers, while the smallest observed are ca. 0.4 µm in diameter. Their shapes are similar. We assigned these plates to the aggregates of the smaller carbonaceous particles. A similar carbonaceous material was prepared in the study,²⁹ and the authors concluded that the material consisted of regularly shaped particles with an average size between 20 and 40 nm. However, such assignment is inconsistent with the luminescence of the CD. It has been shown that the luminescence under UV light is a specific feature of the carbonaceous nanoparticles, which have a size of 1–10 nm (carbon quantum dots).⁴⁶ The color of the luminescence is a complex function of size and surface groups of the carbon quantum dots, but on the grounds of general tendencies, we suppose that the material, prepared in this study, contains CD that are about 2 nm in size (supposedly ±1 nm).⁴⁷ This conclusion does not preclude the presence of larger particles in the sample.

There were two strong spectral bands in the UV spectra of the solutions of CD (Fig. 4a). The first peak appears at λ_max = 213 nm, and it can be attributed to the π–π* transitions of the aromatic –C C– bonds of sp² hybridized domains of the graphite core. The second peak at λ_max = 280 nm can be attributed to the n–π* transition of –C O groups.⁴⁸ The intensity of these two bands in the UV spectrum of the CD is very close. These spectra are very similar to those observed previously.²⁹ Notably, in the case of quantum dots, synthesized by a hydrothermal method starting from glutamic acid and m-phenylenediamine, a wide band located between 250 and 300 nm was assigned to the π–π* transitions of aromatic –C C– bonds, and a much weaker band observed at λ_max = 450 nm was assigned to the –C O groups.⁴⁹

Fig. 4. UV spectrum of the solutions of CD in water (a). IR spectra of the solid CD and PB in KBr pellets (b).

The IR spectrum of the CD was measured for the dried residue of the colloidal solution. There are several strong bands in the spectra (Fig. 4b), which can be assigned to the vibrations of the hydroxyl and amino-groups (3250 cm⁻¹), stretching vibrations of C O and C C groups (1690–1645 cm⁻¹) and bending vibrations of C–H or symmetric vibration of carboxy-groups (1430–1405 cm⁻¹). In addition, the peak at 1200 cm⁻¹ can be attributed to the stretching vibration of C–O of the carboxyl groups, and a peak at 1110 cm⁻¹ to the stretching vibration of C–OH.⁵⁰ The peak at 790 cm⁻¹ may be assigned to out-of-plane C–H,⁴⁹ and an intense peak at 504 cm⁻¹ may be assigned to the stretching vibration of C–C bonds.⁵¹ The presence of all such groups is typical for the CD, obtained by the hydrothermal method from biowaste.^44,50 These bands were found in the IR spectrum of the PB sample at essentially the same wavenumbers (Fig. 4b).²⁰ Notably, the bands in the FTIR spectrum of the CD were significantly wider than the bands in the FTIR spectrum of the PB. This difference may be explained by the presence of a larger quantity of similar but structurally non-equivalent functional groups in the CD; similar wide bands in the IR spectra of CD have been reported previously.^52,53

Finally, the UV and IR spectra of the CD are consistent with the presence of a large quantity of functional groups in this material (which are predominantly oxygen-containing, as it can be concluded from EDX analysis).

3.2. Animal experiments using radiolabelled and fluorimetric approaches: comparative assessment of the neurotropic activity of the PB, CD and SP

The extrasynaptosomal levels of key inhibitory and excitatory neurotransmitters [³H]GABA and L-[³H]glutamate are crucial synaptic characteristics that reproduce plasma membrane integrity and membrane potential availability, which in turn determine transporter-mediated uptake and release.^54,55 It was demonstrated in comparative experiments that the PB did not influence the extrasynaptosomal levels of [³H]GABA and L-[³H]glutamate at a concentration of 0.1 mg mL⁻¹ (Fig. 5). A 10-time increase in the concentration of the PB up to 1.0 mg mL⁻¹ was not accompanied by subsequent elevation of the extrasynaptosomal levels of [³H]GABA and L-[³H]glutamate (Fig. 5). Therefore, the PB was biocompatible and did not possess neurotoxic signs within the concentration range of 0.1–1.0 mg mL⁻¹.

Fig. 5. Extrasynaptosomal levels of [³H]GABA (a) and L-[³H]glutamate (b) in the control and in the presence of the PB, CD and SP at concentrations of 0.1–1.0 mg mL⁻¹. The data are expressed as mean ± SEM. **p < 0.01 and ***p < 0.001 as compared with the appropriate control; n = 12.

In contrast, the CD increased the extrasynaptosomal levels of both [³H]GABA and L-[³H]glutamate starting from a concentration of 0.5 mg mL⁻¹ and SP starting from 0.1 mg mL⁻¹ (Fig. 5).

Therefore, the PB was biocompatible and did not possess neurotoxic signs at a concentration less than 1.0 mg mL⁻¹, while the CD and SP caused neurotoxic consequences at the presynaptic level at concentrations less than 0.5 and 0.1 mg mL⁻¹, respectively.

The radiolabelled results were cross-checked using fluorimetry. Direct measurements of the membrane potential of the nerve terminals in the presence of PB using the potential-sensitive fluorescent dye rhodamine 6G was impossible because of the direct interaction of the PB with the dye. Our recent study has revealed that the JC-1-based approach allowed measuring the mitochondrial membrane potential of nerve terminals directly in the presence of the biochar.²⁰

It was shown that the PB did not change the mitochondrial membrane potential of nerve terminals, while both the CD and SP significantly depolarized the mitochondrial membrane of nerve terminals by 62% and 68%, respectively. Therefore, the effects of the PB, on the one side, and those of the CD and SP, on the other side, on the mitochondrial membrane potential of nerve terminals were opposite. Therefore, the radiolabelled results were confirmed using fluorimetry.

3.3. Comparative assessment of the capability of the PB, CD and SP to mitigate Cu²⁺-induced toxicity in the nerve terminal model using radiolabelled and fluorimetric approaches

3.3.1. Extrasynaptosomal levels of GABA and glutamate measured using radiolabelled neurotransmitters

As shown in Fig. 6, the PB efficiently decreased the Cu²⁺-induced increase in the extrasynaptosomal levels of [³H]GABA (Fig. 6a) and L-[³H]glutamate (Fig. 6b). The addition of the mix of Cu²⁺ and PB after their preliminary incubation for 30 min to the nerve terminals significantly eliminated Cu²⁺-induced toxic effects on the extracellular levels of both [³H]GABA (Fig. 6a) and L-[³H]glutamate (Fig. 6b). Notably, the preliminary interaction/adsorption of Cu²⁺ and PB in water was strong enough to be preserved in the synaptosomal incubation media (Fig. 6a and b).

Fig. 6. Extrasynaptosomal levels of [³H]GABA (a) and L-[³H]glutamate (b) in the nerve terminal preparations in the control and in the presence of the PB (1.0 mg mL⁻¹), CD (1.0 mg mL⁻¹), SP (0.1 mg mL⁻¹) and CuSO₄ (50 µM). The data are expressed as mean ± SEM. **p < 0.01, ***p < 0.001, and n.s.: no significant differences as compared with the appropriate control; ###p < 0.001 as compared with the Cu²⁺ effect; and n = 12.

In order to compare the effects of PB with those of CD and SP in nerve terminals, their capability to mitigate Cu²⁺-induced toxicity was assessed using a similar methodological approach and equal concentrations. It was revealed that the CD amplified the Cu²⁺-induced increase in the extrasynaptosomal levels of both neurotransmitters [³H]GABA (a) and L-[³H]glutamate (b) in a synergistic manner (Fig. 6). The addition of the mix of Cu²⁺ and the CD after their preliminary incubation for 30 min to the nerve terminals increased Cu²⁺ effects on the extrasynaptosomal level of [³H]GABA (Fig. 6a) and L-[³H]glutamate (Fig. 6b) by 9.6% and 13.9%, respectively.

Similar experiments were conducted with SP preparations. It was revealed that SP magnified the Cu²⁺-induced increase in the extrasynaptosomal levels of [³H]GABA (a) and L-[³H] glutamate (b) in a synergistic manner (Fig. 6). The addition of the mix of Cu²⁺ and SP after their preliminary incubation for 30 min to the nerve terminals increased the Cu²⁺-induced effects on the extrasynaptosomal level of [³H]GABA (Fig. 6a) and L-[³H]glutamate by 28.4% and 31.5%, respectively (Fig. 6b).

All the experimental data were analyzed by two-way ANOVA followed by Tukey's post hoc test using the PB/CD/SP and Cu²⁺ as independent factors. Two-way ANOVA revealed a significant interaction between PB and Cu²⁺ [F_(1,44) = 634; p < 0.001; n = 12], CD and Cu²⁺ [F_(1,44) = 7.6; p < 0.01; n = 12], and SP and Cu²⁺ [F_(1,44) = 56.4; p < 0.001; n = 12] in [³H]GABA experiments and a significant interaction between PB and Cu²⁺ [F_(1,44) = 1184; p < 0.001; n = 12], CD and Cu²⁺ [F_(1,44) = 12.4; p < 0.001; n = 12], and SP and Cu²⁺ [F_(1,44) = 88.4; p < 0.001; n = 12] in L-[ ³H]glutamate experiments.

Therefore, the effects of PB, on the one side, and those of the CD and SP, on the other side, on the Cu²⁺-induced neurotoxicity were opposite.

3.3.2. Plasma membrane potential of nerve terminals measured using fluorescent dye rhodamine 6G

To analyze the effects of the PB on Cu²⁺-induced depolarization of the plasma membrane in nerve terminals, the methodological approach avoiding direct interaction of the dye with the biochar was applied. This included preliminary incubation of carbonaceous materials with Cu²⁺ in aquatic media for 30 min followed by centrifugation, where the obtained supernatant was added to the nerve terminals preloaded with rhodamine 6G.

As shown in Fig. 7a, PB almost completely reduced Cu²⁺-induced depolarization of the plasma membrane of nerve terminals. In contrast, the CD and SP further increased Cu²⁺-induced membrane depolarization in a synergistic manner, thereby confirming the radiolabelled experimental data (Fig. 7).

Fig. 7. Plasma membrane potential of the nerve terminals measured using the rhodamine 6G dye. Effects of the PB (1.0 mg mL⁻¹) (a) and CD (0.1 mg mL⁻¹) and SP (0.1 mg mL⁻¹) (b) on the Cu²⁺-induced depolarization of the plasma membrane in nerve terminals.

Therefore, the effects of the PB, on the one side, and those of the CD and SP, on the other side, on the Cu²⁺-induced depolarization of the plasma membrane of nerve terminals were opposite. The PB mitigated Cu²⁺-induced plasma membrane depolarization, while the CD and SP demonstrated cumulative effects with Cu²⁺, further depolarizing the plasma membrane of nerve terminals.

3.3.3. Mitochondrial membrane potential of nerve terminals measured using the fluorescent dye JC-1

As shown in Fig. 8, the PB reduced the Cu²⁺-induced mitochondrial membrane depolarization of nerve terminals significantly. In contrast, it was shown that both the CD and SP further increased the Cu²⁺-induced depolarization of the mitochondrial membrane of nerve terminals, thereby confirming the above-mentioned experimental data (Fig. 8).

Fig. 8. Mitochondrial membrane potential of the nerve terminals measured using the JC-1 dye. Effects of the PB, CD and SP on the Cu²⁺-induced mitochondrial membrane depolarization of nerve terminals. The ratio of the fluorescence intensity at 590 vs. 530 nm. The dye (5 µM) was added to the synaptosome suspension (0.15 mg of protein per mL) and incubated in the dark at a temperature of 37 °C for 10 min. Then, Cu²⁺ (50 µM CuSO₄), PB (1.0 mg mL⁻¹), CD (0.1 mg mL⁻¹), SP (0.1 mg mL⁻¹) or premix (mixed and preincubated for 30 min) Cu²⁺ + PB/CD/SP aliquots were added to the cuvette, and the dye fluorescence was monitored at 20 min time points at an excitation wavelength of 485 nm and an emission wavelength ranging from 510 to 610 nm. The data are expressed as mean ± SEM. ***p < 0.001; n.s.: no significant differences as compared with the control; ###p < 0.001; as compared with the Cu²⁺effect; n = 12.

Two-way ANOVA revealed a significant interaction between the PB and Cu²⁺ [F_(1,44) = 57.1; p < 0.001; n = 12], CD and Cu ²⁺ [F_(1,44) = 20.3; p < 0.001; n = 12], and SP and Cu²⁺ [F_(1,44) = 28.6; p < 0.001; n = 12].

Therefore, the effects of the PB, on the one hand, and those of the CD and SP, on the other hand, on the Cu²⁺-induced mitochondrial membrane depolarization were opposite. The PB alleviated Cu²⁺-induced mitochondrial membrane depolarization, while the CD and SP further depolarized the mitochondrial membrane of nerve terminals.

3.4. Comparative assessment of the capacity of the PB, CD and SP to influence spontaneous and stimulated ROS generation in nerve terminals

Spontaneous and stimulated ROS generation in nerve terminals was analysed using the fluorescent dye DCF. In our recent study, it has been shown that the PB per se interacted with the dye.²⁰ In this context, the following DCF experiments in nerve terminals were conducted using the CD and SP. As shown in Fig. 9a and b, the CD alone decreased spontaneous ROS generation in nerve terminals, and so demonstrated strong antioxidant-like properties, in contrast to Cu²⁺, which stimulated ROS generation in synaptosomes upon addition (Fig. 9a and b). However, when the CD was pre-incubated with Cu²⁺, this mixture significantly potentiated the effect of Cu²⁺. The same effect was also observed for SP, as can be seen in Fig. 9c and d, where the addition of the mixture of SP and Cu²⁺ to synaptosomes enhanced the effect of Cu²⁺, at least for the first 2–3 min (Fig. 9d; calculation for 3-min time points), while SP alone significantly decreased spontaneous ROS generation.

Fig. 9. Effects of the CD (a and b) and SP (c and d) on the spontaneous and Cu²⁺-induced ROS generation in the nerve terminals. CD (50 µg mL⁻¹), SP (45 µg mL⁻¹) and their mixtures with CuSO₄ (50 µM), preliminarily incubated for 15–30 min at room temperature, added to synaptosomes preincubated with H2-DCFDA (5 µM). (e and f) For comparison, the effect of the CD on Hg²⁺-induced ROS generation: CD (6 µg mL⁻¹), HgCl₂ (5 µM) and their mixture added to synaptosomes, as indicated by the arrow. The traces represent the data from 12 experiments performed with different preparations. (b, d and f) Calculated levels of the DCF fluorescence signal intensity at 700-, 380- and 700-s time points, respectively, in response to the application of Cu²⁺/Hg²⁺per se and CD/SP per se and combined application of Cu²⁺/Hg²⁺ + CD/SP. The data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: no significant differences as compared with the appropriate control; #p < 0.05; ###p < 0.001; as compared with the Cu²⁺/Hg²⁺ effect; n = 12.

In additional experiments, we analysed the capacity of the CD to influence ROS generation induced by the other heavy metal Hg²⁺. As shown in Fig. 9i and f, Hg²⁺ at a low concentration of 5 µM induced significant ROS generation in synaptosomal incubation media. In contrast to the above-mentioned experiments with Cu²⁺, the changes in DCF fluorescence registered during combined administration of both the CD and Hg²⁺ were opposite to those induced by Hg²⁺ alone, thereby indicating the efficacy of the CD to reduce the harmful effect of Hg²⁺ (explanation is provided below in the Discussion section). Notably, the concentration of Hg²⁺ used in the experiments was an order lower than that of Cu²⁺ to produce comparable effects.

The experimental data were analyzed by two-way ANOVA followed by Tukey's post hoc test using the CD/SP and Cu²⁺/Hg²⁺ as independent factors. Two-way ANOVA revealed a significant interaction between the CD and Cu²⁺ [F_(1,44) = 33.3; p < 0.001; n = 12], SP and Cu²⁺ [F_(1,44) = 6.79; p < 0.05; n = 12], and the CD and Hg²⁺ [F_(1,44) = 18.2; p < 0.001; n = 12].

Interestingly, the effect of SP on Cu²⁺-induced ROS generation was very similar to that recently reported for a wood smoke particulate matter preparation.³³ In both cases, the simultaneous presence of Cu²⁺ and smoke preparations from wood or corn cob waste potentiated the effect of Cu²⁺.

4. Discussion

The conversion of agricultural waste into biochars and other carbon nanomaterials represents an efficient strategy for waste valorization that goes in line with the circular economy. The global concern regarding agricultural waste management, particularly corn cobs, necessitates the development of “green” valorization strategies.^56–59 In this study, “green” carbonaceous nanoparticles/materials, such as PB, CD, and SP, were compared regarding their biocompatibility and interaction with heavy metals, i.e. Cu²⁺, in the nerve terminal model. In this series of materials, the PB and CD have a large content of the functional groups. It was revealed that the form of carbonaceous materials and the resulting physicochemical properties fundamentally determined both the characteristics.

A critical finding of this study is the high biocompatibility of the PB compared to the CD and SP. In a synaptosome model, the PB did not perturb neither the extrasynaptosomal levels of [³H]GABA and L-[³H]glutamate nor the mitochondrial membrane potential (Fig. 5 and 8). This result was consistent with the reports, indicating that biochars exhibited low cytotoxicity and minimal biological reactivity due to their relatively large particle size, limited cellular uptake, and stable surface chemistry.^10,15 This stability is likely attributed to the high-temperature pyrolysis, which produces a more chemically inert, carbon-rich framework with a reduced volatile organic content.⁶⁰

Conversely, the CD significantly increased the extrasynaptosomal [³H]GABA and L-[³H]glutamate levels and induced mitochondrial membrane depolarization (Fig. 5 and 8). Synaptic mitochondria are particularly vulnerable to physicochemical stressors, and their dysfunction can lead to impaired neurotransmitter cycling, reduced ATP production, and enhanced ROS generation. It was demonstrated in the literature data that nanomaterials, including various carbon-based nanoparticles, can induce mitochondrial depolarization and oxidative stress in neuronal cells.⁶¹ Carbon dots, despite their generally reported low cytotoxicity and high biocompatibility in various cell models, are nanoscale particles with high surface reactivity and the capability to interact with biological membranes and intracellular organelles. Recent studies on biomass-derived carbon dots have suggested that their surface-active groups (e.g., carboxyl and amino groups from hydrothermal synthesis) can interact with presynaptic membrane proteins or lipid bilayers, leading to depolarization.^23,62,63 In our previous study, carbon dots from β-alanine enhanced the extrasynaptosomal levels of excitatory neurotransmitters L-[¹⁴C]glutamate and D-[2,3-³H]aspartate and inhibitory neurotransmitters [³H]GABA and [¹⁴C]glycine and simultaneously increased the conductance of the artificial planar lipid bilayer by inducing stable potential-dependent cation-selective pores, thereby confirming the primary role of lipids in carbon dot-cell membrane interaction.⁶⁴ The present findings extend these observations at the presynaptic level and emphasize that “green” synthesis alone does not guarantee neurobiological safety.

Similarly to the CD, SP also significantly increased the extrasynaptosomal neurotransmitter levels and induced mitochondrial membrane depolarization (Fig. 5 and 8). Previously, we have demonstrated that SP obtained from wood and plastics inhibited transporter-mediated uptake of L-[¹⁴C]glutamate and [³H] GABA in nerve terminals, thereby increasing their ambient extracellular concentrations, which is a hallmark of excitotoxicity and disturbance of excitation-inhibition balance.^27,31,32 Combustion-derived particulate matter contained heterogeneous carbon structures along with adsorbed organic compounds and trace metals, which can enhance oxidative stress and membrane–disruptive properties.⁶⁵

An important finding of this study is the different capacity of the tested carbonaceous nanoparticles/materials to modulate Cu²⁺-induced neurotoxicity (Fig. 6–8). Cu²⁺ is an essential trace element involved in numerous enzymatic processes, but its excessive concentrations promote the transformation of Cu²⁺ to a potent neurotoxin that disrupts mitochondrial function and synaptic signaling, triggers ROS generation, and promotes lipid peroxidation and protein oxidation in neuronal systems, contributing to neuronal damage and neurodegenerative processes.^66,67 Cu pollution raises a global environmental concern, and widespread Cu pollution results in excessive copper exposure in humans. Cu is mostly absorbed by humans through diet and environmental or occupational exposure.⁶⁸ Cu uptake occurs mainly through the small intestine, and the small intestine epithelium took up copper ions via copper transporters. Recently, it has been suggested that cuproptosis is an independent form of cell death and is closely associated with impaired mitochondrial respiration and lipoic acid pathway.^69,70

Comparative analysis of different carbonaceous materials using several methods (radiolabeled and fluorescent ones) has many complications that need to be overcome. Carbonaceous materials showed different colors, transparency in the synaptosomal incubation media, interaction with fluorescent dyes, and basal neurotoxicity (Fig. 5). In particular, the fluorescence intensities of the JC-1 and DCF dyes were recorded inside synaptosomes, while rhodamine 6G can directly interact with the carbonaceous materials in the synaptosome incubation media. Such interaction of rhodamine 6G and JC-1 with pyrolysis biochars was demonstrated in our previous work.^20,71 Despite unspecific interaction of JC-1 with biochars, the mitochondrial membrane potential can be measured directly in the presence of biochars in the synaptosome incubation media, because the calculations were based on the ratio of JC-1 fluorescence at different wavelengths.²⁰ These calculations of the fluorescence intensity at 590 vs. 530 nm allowed disregarding the difference in the JC-1 loading by synaptosomes, the number of mitochondria and the probe fluorescence quenching. In a control DCF fluorescence quenching test in the absence of synaptosomes using the commercially available oxidized product DCF, it was shown that the addition of the CD and SP to DCF in Krebs–Ringer buffer did not alter the probe fluorescence, indicating the absence of any detectable optical interference or fluorescence quenching under these conditions (unpublished data). From biological and potential medical application point of view, the comparative series of the experiments on Cu²⁺-induced toxicity modulation were carried out using maximal “safe” concentrations of carbonaceous materials (Fig. 5) in order to avoid bio-incompatibility during their implementation in medical practice in the future. From experimental point of view, definite concentrations were selected for each tested carbonaceous material to avoid inaccuracy in the registration of potential additive, synergistic or inhibiting mode of their action with Cu²⁺. In particular, SP at a concentration of 1 mg mL⁻¹ (Fig. 5) induced almost complete leakage of radioactive neurotransmitters accumulated by synaptosomes that made inaccurate further experiments on combined effects with Cu²⁺ at this SP concentration. Our results demonstrated that the PB significantly mitigated Cu²⁺-induced membrane depolarization and neurotransmitter leakage (Fig. 6–8). Biochar materials obtained by pyrolysis typically possess a highly developed porous structure and abundant oxygen-containing functional groups that facilitate the binding of heavy metals through electrostatic interactions, ion exchange, and surface complexation mechanisms.^72,73 The protective effect of the PB demonstrated in this study can be explained by the high specific surface area and the presence of oxygen-containing functional groups (carbonyl and aliphatic groups) on the biochar surface, which facilitate the adsorption and immobilization of Cu²⁺.^19,60 By sequestering Cu²⁺, the PB reduced the bioavailability of the metal, preventing its direct interaction with the synaptosomal plasma and mitochondrial membranes. This result is in line with the well-documented capacity of biochars to adsorb heavy metals in non-biological systems.^15,17 Numerous recent studies have confirmed the high efficiency of biochar materials in Cu removal from aqueous environments. For example, modified biochar composites demonstrated Cu²⁺ removal efficiencies exceeding 90% due to electrostatic interactions, surface complexation, and precipitation mechanisms. Similarly, biochar-based adsorbents prepared from different biomass sources exhibited adsorption capacities above 25–30 mg g⁻¹ for Cu²⁺ ions in aqueous solutions.^19,59 In addition, advanced biochar composites incorporating chitosan or magnetic nanoparticles can further enhance the Cu removal efficiency through synergistic adsorption mechanisms.⁵⁹ The biochar has been widely reported as an effective material for heavy-metal immobilization due to its high surface area, aromatic carbon structure, and tunable surface chemistry.^72,73

In contrast to the biochar, the CD and SP acted synergistically with Cu²⁺, exacerbating neurotoxicity (Fig. 6–8), thereby representing a “multicomponent hazard”. This paradoxical effect may be attributed to several factors. First, nanoscale particles may facilitate metal ion transport toward biological membranes or intracellular compartments, effectively increasing the local Cu²⁺ concentrations at sensitive synaptic targets. This suggestion is supported by the data on the efficient binding of Cu²⁺ by the carbon dots, bearing different functional groups on the surface.^52,53 It was reported that the sorption of Cu²⁺ by CD was facilitated by the presence of a large quantity of functional groups on the surface (carboxy, hydroxy, and amino-groups), and the interaction of Cu²⁺ with N-doped CD led to their aggregation,⁵² implying that the CD acted as chelators, which coordinate to one metal ion filling multiple coordination sites. Second, the interaction of Cu²⁺ with reactive surface groups on the CD or with redox-active components of SP could promote ROS formation via Fenton-like reactions, thereby amplifying oxidative damage. Since Cu²⁺ catalyzed redox cycling and targeted mitochondrial networks in neuronal systems,^66,74 its combined presence with reactive carbon nanoparticles may synergistically enhance neurotoxicity. When Cu²⁺ was combined with the CD or SP, which independently causes membrane depolarization, the cumulative stress on the synaptic machinery led to a catastrophic failure of membrane potential maintenance. Our findings align with our recent evidence, showing that carbonaceous nanoparticles can modulate metal-induced excitotoxicity, often shifting the balance toward increased ROS generation and synergistic membrane depolarization.^33,75

The different effects of the CD on ROS generation induced by Cu²⁺ and Hg²⁺ in the synaptosomal model shown in additional experiments (Fig. 9) may reflect the fundamental differences in the redox chemistry and cellular mechanisms of toxicity of these metals. Cu²⁺ is a redox-active transition metal capable of cycling between Cu²⁺ and Cu⁺ oxidation states and catalyzing Fenton-like reactions that generate highly reactive hydroxyl radicals.^76,77 Even when partially adsorbed onto carbonaceous surfaces, Cu²⁺ may retain redox activity or participate in surface-mediated electron transfer reactions, which can sustain ROS formation. In addition, interactions between transition metals and carbon-based nanomaterials may facilitate electron transfer processes on the nanoparticle surface, potentially maintaining the generation of ROS.⁷⁸ Therefore, the presence of the CD may not effectively suppress Cu²⁺-driven oxidative reactions in synaptosomes, which could explain why the CD did not significantly attenuate Cu²⁺-induced ROS generation in the present study (Fig. 9). In contrast, Hg²⁺ is not a redox-active metal under physiological conditions and induces oxidative stress mainly through indirect mechanisms. Hg²⁺ have a strong affinity for sulfhydryl groups in proteins, which leads to the inhibition of antioxidant systems such as glutathione-dependent enzymes and thioredoxin systems.^79–81 The sequestration of Hg²⁺ by carbon nanoparticles may effectively decrease the pool of bioavailable metal ions and prevent their interaction with thiol-containing proteins and enzymes in synaptosomes. Consequently, the binding of Hg²⁺ to the CD could reduce Hg²⁺-induced oxidative stress and attenuate ROS generation in nerve terminals. In addition, the CD possesses surface functional groups capable of electron donation and radical scavenging, which may further suppress ROS generation through indirect mechanisms of toxicity.^82,83 The difference in effective metal concentrations used in the present experiments may also contribute to the observed phenomenon. Hg²⁺ induced measurable ROS generation at a concentration of 5 µM, while Cu²⁺ required a ten-fold higher concentration (50 µM) to produce reproducible oxidative effects. Under such conditions, the binding capacity of the CD may be sufficient to partially neutralize Hg²⁺ ions but insufficient to sequester the larger amount of Cu²⁺ present in the incubation medium.⁷⁸

5. Conclusion

Overall, the comparative analysis demonstrated that carbonaceous nanoparticles/materials derived from corn cob waste exhibited markedly different biocompatibility profiles and opposite effects on Cu²⁺-induced neurotoxicity. The divergent effects observed between the PB and the other two materials underscore the importance of “safe-by-design” principles in the valorization of agricultural waste. While hydrothermal synthesis (CD) and open burning smoke collection (SP) are technically “green” or simple approaches, they may yield materials with inherent bioactivity that compromises their safety in biological environments. Transforming corn cob waste into the pyrolytic biochar (PB), however, shows a dual benefit: it effectively converts corn cob waste into functional carbon materials while mitigating environmental and health risks, providing a safe, neuroprotective material capable of mitigating Cu²⁺ toxicity.

From an environmental perspective, the results underscore that not all carbonaceous materials derived from agricultural waste are equally safe for biological systems, even when produced using “green” methodologies. While the PB appears suitable for applications involving heavy metal sequestration with minimal neurobiological risks, CD and SP require careful toxicological evaluation before being considered for environmental or biomedical uses. The enhanced neurotoxicity observed in the presence of Cu²⁺ is particularly relevant to polluted environments, where multiple contaminants coexist.

The vast global availability of corn cob waste, combined with its favourable chemical composition, positions it as a highly attractive renewable resource for the development of biomedical nanomaterials. Valorization pathways leading to green-synthesized biochars not only address the environmental problem of corn cob disposal, but also enable the production of advanced materials for medical and healthcare applications.^5–9 The PB due to its unique ability to eliminate harmful Cu²⁺-induced effects in the nerve terminal model may have potential for application in Cu²⁺-targeted biotechnologies and medicine, in particular in the treatment of Wilson's disease and environmental Cu²⁺-overload in humans. However, due to the limitation of the in vitro synaptosomal model used in this study, further detailed in vivo exposure experiments using rodents are necessary to support biomedical translational claims.

Although isolated synaptosomes provide a sensitive and mechanistically informative model of presynaptic function, extrapolation to in vivo systems should be made. Future studies should address long-term exposure, nanoparticle/material internalization pathways, and the role of protein corona formation in modulating nanoparticle–neuron interactions. Additionally, systematic characterization of the surface chemistry and metal-binding mechanisms of each nanoparticle and material will be essential to optimize the balance between the adsorption capacity and the neurobiological safety. Further studies are needed to clarify the relationship between the structural characteristics of biochars (surface functional groups, pore size distribution, and aromaticity) and their neuroprotective potential.

Ethical approval

Animal-involved experiments were performed according to the “Scientific Requirements and Research Protocols” and “Research Ethics Committees” of the Declaration of Helsinki and the “ARRIVE guidelines for reporting experiments involving animal”^34,35 and approved by the Institutional Animal Care and Use Committee (Protocol No. 1, dated 15 January 2025).

Author contributions

Nataliya Krisanova: conceptualization, project administration, funding acquisition, methodology, investigation, data curation, formal analysis, writing – review and editing, writing – original draft; Anastasiya Terebilenko: methodology, investigation, data curation, formal analysis; Natalia Pozdnyakova: methodology, investigation, data curation, formal analysis; Alla Tarasenko: methodology, investigation; data curation, formal analysis; Marina Dudarenko: methodology, investigation; Arsenii Borysov: methodology, investigation; Roman Sivko: methodology, investigation; Yaroslav Kurys: investigation, data curation, formal analysis; Denys Mazur: methodology, investigation, data curation, formal analysis; Tatiana Borisova: data curation, formal analysis, writing – review and editing, writing – original draft; Sergey V. Kolotilov: data curation, formal analysis, writing – review and editing, writing – original draft. PB and CD synthesis and characterization: AT, YK, DM, SK; synaptosome preparations: RS, MD; L-[³H]glutamate and [³H]GABA experiments: NK, MD, NP; fluorimetry: AT, NK, AB, RS; data analysis and figure preparation: NP, NK, YK, DM, TB, SK; funding acquisitions, project leading: NK; data analysis and paper writing: NK, TB, SK.

Conflicts of interest

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

Data availability

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