Disposable facemask waste combustion emits neuroactive smoke particulate matter

Tremendous deposits of disposable medical facemask waste after the COVID-19 pandemic require improvement of waste management practice according to WHO report 2022, moreover facemasks are still in use around the world to protect against numerous airborne infections. Here, water-suspended smoke preparations from the combustion of disposable medical facemasks (polypropylene fibers) were collected; size, zeta potential, surface groups of smoke particulate matter were determined by dynamic light scattering, FTIR and Raman spectroscopy, and their optical properties were characterized. Neurochemical study using nerve terminals isolated from rat cortex revealed a significant decrease in the initial rate of the uptake/accumulation of excitatory and inhibitory neurotransmitters, L-[14C]glutamate and [3H]GABA, and exocytotic release, and also an increase in the extracellular level of these neurotransmitters. Fluorescent measurements revealed that ROS generation induced by hydrogen peroxide and glutamate receptor agonist kainate decreased in nerve terminals. A decrease in the membrane potential of nerve terminals and isolated neurons, the mitochondrial potential and synaptic vesicle acidification was also shown. Therefore, accidental or intentional utilization of disposable medical facemask waste by combustion results in the release of neuroactive ultrafine particulate matter to the environment, thereby contributing to plastic-associated pollution of air and water resources and neuropathology development and expansion.

pandemic, the facemasks are still used by the population around the world in daily life in order to protect against numerous airborne infections.Disposability of the facemasks reduces the contamination risks as compared to the reusable ones [8][9][10] .
According to WHO report 2022, thousands of tonnes of extra medical waste from the COVID-19 pandemic has put tremendous strain on waste management systems worldwide, threatening environment and human health and underlying critical needs to improve waste management practices 11 .Huge amount of used facemasks requires establishment of disposal routes to reduce the harmful effects of their waste on the environment 7 .30% of healthcare facilities in developed countries and 60% in the least developed ones are not equipped to handle existing waste loads and moreover the additional COVID-19 load.Current literature shows that the huge number of contaminated facemasks is a problem for the environment, as the current processes of disposal (i.e.incineration and reclamation) are accompanied by the release of toxic chemicals 12,13 .Poorly managed landfills and waste disposal sites potentially impact communities through contaminated air from burning waste, and poor water quality 11 .Accidental burning of used facemasks can also occur during fires of the domestic mixed facemask-containing garbage, and besides that intentional uncontrolled illegal utilisation of the facemask waste by combustion is still of practice in the least developed countries.
There are plenty of biopolymers available in the market, whereas most disposable single-use facemasks are made from fossil-derived polymers 14,15 .The standard textile used to mass-produce the disposable medical facemasks is polypropylene sheets that are made using spun-bond or melt-blown technology 16 .Material recycling of used facemask waste is impossible, as they can be contaminated with pathogens and have to be considered and handled as hazardous waste, and so thermal utilization offers a reliable disposal route for used facemasks [8][9][10] .
Under laboratory conditions, we have recently synthesized the water-suspended smoke samples from plastics that are appropriate for neurochemical testing using rat brain nerve terminals.These preparations contained major nano-sized (30 nm) PM fraction and impaired functioning of neurotransmitter transporters, thereby provoking presynaptic malfunctioning and triggering neurological consequences 17 .It should be underlined that impairment of transport of excitatory and inhibitory neurotransmitters in the central nervous system, glutamate and γ-aminobutyric acid (GABA), respectively, is involved in the pathogenesis of major neurological complications and disorders.Also, a comparative neurochemical study of wood sawdust and plastic smoke particulate matter preparations revealed higher riskiness in mitigation of synaptic inhibition by plastic preparations as compared to the wood sawdust ones 18 .
Taking into account a high significance and globalism of disposable facemask waste utilization problem, the aims of this study were: (1) to collect in the laboratory conditions water-suspended smoke PM preparations of disposable medical facemasks from polypropylene fibers (mask preparations (MPs)) using previously developed methodical approach 17 ; (2) to define the average size and Z-potential of particles in MPs using dynamic light scattering; (3) to analyse optical, fluorescence and surface properties of particles in MPs using Raman and Fourier-transform infrared (FTIR) spectra; (4) to assess neurotoxic properties of MPs studying key characteristics of synaptic neurotransmission in isolated rat cortex nerve terminals (synaptosomes), namely Na + -dependent transporter-mediated uptake and release, exocytosis and the ambient levels of L-[ 14 C]glutamate and [ 3 H]GABA; (5) to analyze MP effects on the membrane potential of synaptosomes and isolated cortex neurons, mitochondrial potential, synaptic vesicle acidification and stimulated generation of reactive oxygen species (ROS) using fluorescent dyes rhodamine 6G, mitochondrial membrane potential assay kit JC-1, acridine orange and 2′,7′-dichlorofluorescein (DCF), respectively.

Dynamic light scatter
MPs were a colloid obtained by filtering through a glass microfiber filter with a pore diameter of 1.0 µm to eliminate debris and enrich preparation with nanoscale fractions.MP colloid was a heterogeneous system with a high index of polydispersity (mean value of 5 measurements was 0.8); the samples contained particles of different hydrodynamic sizes.Figure 1a shows particle size distribution by intensity as a direct value.Figure 1b represents particle distribution by number, and several populations of nanoparticles of different size were observed, in particular MPs contained four fractions of a wide nano-sized range with mean values of hydrodynamic diameter equaled to 396 nm, 190 nm, 10 nm and 1.2 nm.Distribution by number showed a relatively equal quantity of particles in each population, with slightly increased content of ultrasmall particles.It differed from particle size distribution by intensity due to the impact of large particles in signal formation on the detector.Z-potential, i.e.net charge of a nanoparticles' surrounding in solvent, was assessed in the next series of the experiments that can indicate a stability of nanoparticles in the colloid.Z-potential of MPs under stock acidic pH 3.75 was equal to − 9.2 ± 5.4 mV (Fig. 1c).So, particles in this colloid had very low net charge that in turn could lead to their coagulation and aggregation in acidic media.Z-potential of MPs was measured also in basic medium at pH 10.1 and had a value of − 24.0 ± 4.9 mV.It means that the system contained carbon nanoparticles with negatively-charged chemical groups at the surface and become more stable at higher pH values.

FTIR and Raman spectroscopy
The spectrum of MPs indicated the presence of two products, i.e. short-chain alcohols (R-C-OH) and carboxylic acids (R-COOH) 19 .Analysis of the position of the infrared (IR) absorption bands (Fig. 2a) from left to right revealed the band in the region 3396 cm −1 , which referred to the stretching vibrations of OH 20 , and a set of peaks at 2966, 2932, 2872, 2854 cm −1 , related to CH stretching vibrations of hydrocarbons 20,21 .A characteristic feature was that the CH bands were not in the form of a shoulder along with the OH chain, but looked like practically separated bands.It was difficult to estimate the length of the hydrocarbon chain according to IR spectra, however www.nature.com/scientificreports/ the ratio of intensity of CH 3 at 2966 cm −1 to CH 2 at 2932 cm −1 indicated a greater contribution of CH 2 .It can be assumed that the C-C chain was short, since both CH 3 bands symmetric and asymmetric were pronounced, and the symmetrical CH 2 at 2854 cm −1 was only in the form of a small shoulder 21 .The position of the band C=O at 1711 cm −1 and the broad OH band at 3396 cm −1 were markers of the presence of a carboxyl molecular group, which was a part of saturated carboxylic acids 22,23 .The shoulder at 1766 cm −1 indicated the presence of monomers with hydrogen bonds, and carbonic acid dimers with hydrogen bonds (band at 1711 cm −1 ) 24,25 .The band at 1590 cm −1 , assigned to the molecular group C=C, can be attributed to carbon particles formed during the combustion of organic compounds.Raman spectra complement the information received from FTIR spectra, since different types of vibrations were active in FTIR and Raman (Table 1).In Raman spectroscopy, the spectrum of MPs registered characteristic markers of CH stretching vibrations in the region at 2966, 2930, 2885 cm −1 , as well as a band at 3085 cm −1 , which refers to C-H in C=C-H group in compounds with rings, and the band of CH 2 deformation vibrations near 1457 cm −1 (Fig. 2b).

Optical and fluorescent properties
The optical properties of MPs were characterized by ultraviolet-visible (UV-Vis) absorption and photoluminescent spectroscopy.Absorption of MPs was mainly registered in the UV region (range of 190-300 nm) (Fig. 3).
Table 1.Assignment of the main spectrum bands to molecular groups according to [19][20][21][22][23][24][25][26] (def., deformation vibrations; str, stretching vibration; sh, shoulder; w, weak; m, medium; s, strong; vs, very strong; vvs, super strong; sym, symmetrical; asym, asymmetrical vibrations).The main light absorption has occurred at 194 nm and 228 nm.These wavelengths correspond to absorption of light with high energies (6.4 and 5.45 eV, respectively).Such shift of absorption into high-energy range could be explained by the presence of organic matter with π-π* transitions inside, involving unsaturated sp 2 carbons (e.g., C=C bonds) 27 .There were no or very low number of molecules capable of absorbing lower energy wavelengths.Despite relatively intense absorption, the samples were found to be non-fluorescent.Such properties tended to possess small non-aromatic organic molecules with multiple conjugated double bonds, often containing carbonyl or nitrogen groups in their structure, which contribute to their strong UV absorption (e.g., ketones, aldehydes, polyenes, etc.) 28,29 .

Neuroactive properties of MPs examined using animals and radiolabelled neurotransmitters
The transporter-mediated uptake of L-[ 14 C]glutamate and [ 3 H]GABA by the nerve terminals in the presence of MPs In the first series of neurotoxicological studies, the effects of MPs on the initial rate of the transporter-mediated uptake and accumulation of excitatory and inhibitory neurotransmitters by the nerve terminals were assessed.The transporter-mediated uptake is one of key processes in the presynapse that determines the proper extracellular level of neurotransmitters in the synaptic cleft 30,31 .It was shown that both parameters, i.e. the initial rate of the uptake and accumulation of L-[ 14 C]glutamate for 10 min, were not altered by MPs at a concentration of 10 μg/ml and significantly reduced in a dose-dependent manner at 50 μg/ml and 200 μg/ml MPs (Fig. 4a).The initial rate of the uptake and accumulation of [ 3 H]GABA by nerve terminals were also insignificantly reduced (a tendency to reduce) by MPs at a concentration of 10 μg/ml, whereas these parameters considerably decreased in a dose-dependent manner at 50 μg/ml and 200 μg/ml MPs (Fig. 4b).

The extracellular level of L-[ 14 C]glutamate and [ 3 H]GABA in the nerve terminal preparations in the presence of MPs
The extracellular level of neurotransmitters between the episodes of exocytotic release is a dynamic balance of transporter-mediated uptake/release and the tonic leakage of neurotransmitters.This is a very important synaptic parameter that determines strength of synaptic contacts and tonic neuronal communication; regulates synaptic neurotransmission and is tightly associated with the energetic status and the membrane integrity of nerve terminals 30,31 .As shown in Table 2, the extracellular level of L-[ 14 C]glutamate and [ 3 H]GABA in the nerve terminal preparations was not changed in the presence of MPs at concentrations of 10 μg/ml and 50 μg/ml, whereas significantly increased at 200 μg/ml.These data are in accordance with above L-[ 14 C]glutamate and [ 3 H] GABA uptake results.In particular, the lesser the uptake was, the higher the extracellular level can be registered.

Tonic release of [ 3 H]GABA from the nerve terminals in the presence of MPs
Tonic release of [ 3 H]GABA from the nerve terminals measured during blockage of GABA transporters by selective inhibitor NO-711 reflected the membrane permeability to small molecules.As shown in Table 3, tonic   The membrane potential of the nerve terminals and isolated cortex neurons in the presence of MPs The potential of the plasma membrane of nerve cells is mainly responsible for proper transporter-mediated uptake, the extracellular level and exocytotic release of the neurotransmitters.It corresponds to the membrane integrity and reflects whether or not tested compounds cause membrane depolarization.The membrane potential of the nerve terminals and neurons isolated from the cortex was monitored using a potential-sensitive fluorescent dye rhodamine 6G.After the addition of the dye to the suspension of synaptosomes or neurons, quenching of the dye fluorescence was observed due to partial binding to a negative charge in the inner near membrane space of synaptosomes or neurons.It was shown that MPs at a concentration of 10 μg/ml did not influence both the synaptosomal and neuronal membrane potentials, whereas an increase in the MP concentration up to 50 and 200 μg/ml depolarized the synaptosomal and neuronal membranes in a dose-dependent manner (Fig. 6a,b).

The mitochondrial membrane potential of the nerve terminals in the presence of MPs
In the next series of the experiments, the mitochondrial membrane potential was analyzed in the nerve terminals using a cationic carbocyanine fluorescent dye JC-1 accumulated by mitochondria.Before kinetic measurements and the addition of MPs, JC-1 fluorescence spectra of each synaptosome sample were recorded at 0 min time point.The fluorescence spectra were similar at 0 min time point in all samples.For quantitative analysis, the ratio of the fluorescence intensity at 590 and 530 nm was used.This ratio makes it possible to overcome the following problems, such as the difference in the probe loading, the number of mitochondria, and MP-associated fluorescence quenching.JC-1 fluorescence spectra in the control and in the presence of MPs at a concentration of 50 μg/ml were monitored at a 30 min time point.In the control, the probe spectrum was revealed to be unchanged for 30 min.As shown in Fig. 7a,b, MPs at a concentration of 50 μg/ml caused depolarization of the mitochondria membrane in the nerve terminals.The ratio of the fluorescence intensity at 590 versus 530 nm in the control was 3.99, whereas it has become 1.82 in the presence of MPs that reflected more than a twofold decrease in the mitochondrial membrane potential.

The acidification of synaptic vesicles in the presence of MPs
Taking into account the harmful influence of MPs on the exocytosis shown in Fig. 5, the effect of MPs on synaptic vesicle acidification was analysed using the pH sensitive fluorescence probe acridine orange (Fig. 8).The

ROS generation in the nerve terminals in the presence of MPs
Kinetics of H 2 O 2 -and kainate (agonist of kainate/AMPA type glutamate receptors)-induced ROS generation in the nerve terminals was monitored using fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H 2 -DCFDA).Figure 9a,b represents a dose-dependent inhibiting effect of MPs at different concentrations on

Discussion
With this study, we are contributing towards the urgent and global problem of utilization of disposable medical facemask deposits underlined in WHO report 2022 11 .To overcome the problem of disposable facemask utilization, it is necessary to determine their degradation and flammability characteristics for efficient waste management 12 .Here, data on dynamic light scattering revealed that MPs were heterogeneous and consisted of four populations of nanoparticles.Distribution by number showed high content of ultrasmall particles with negatively-charged chemical groups at the surface (Fig. 1).According to the literature data, ultrafine air pollution www.nature.com/scientificreports/PM 0.1 (less than 0.1 µm in diameter) is more harmful to human health as compared to fine PM 2.5 and coarse PM 10 (less than 10 µm in diameter) ones.The lesser the diameter of air pollution PM is, the higher toxicity via mechanisms of oxidative stress and inflammation can be registered 32 .
In this study, it was demonstrated that MPs decreased the transporter-mediated uptake of L-[ 14 C]glutamate and [ 3 H]GABA, and exocytotic release, as well as increased the extracellular level of both neurotransmitters.MP-induced changes in these characteristics were in accordance with each other and represented experimental "cross-check" parameters for assessment of synaptic neurotransmission.For example, a MP-induced increase in the extracellular level of neurotransmitters (a balance of uptake and tonic release) (Table 2) agreed with decreased synaptosomal neurotransmitter uptake by MPs (Fig. 4).It should be noted that an increase in the extracellular level of glutamate in the nerve terminals above definite individual threshold can cause neurotoxicity and death of neurons via over activation of postsynaptic ionotropic glutamate receptors 30,31 .Also, data on changed neurotransmitter transport in the presence of MPs agreed with our recent studies regarding neurotoxic effects of water-suspended plastic smoke preparations 17,18 , where smoke preparations from polyethylene bottles showed almost similar changes (as compared to MPs) in L-[ 14 C]glutamate and [ 3 H]GABA transport in nerve terminals.
ROS are involved in the development of many human diseases and also perform physiological functions.ROS are counterbalanced by an antioxidant defense system that modulates ROS levels thereby allowing their physiological roles and minimizing the oxidative damage, which in turn provokes disease development 33 .It was suggested that the critical role in the redox balance maintenance belongs to antioxidant enzymes, but not small-molecule antioxidant compounds 34 .Here, it was shown that MPs decreased H 2 O 2 -and kainate-induced ROS generation (Fig. 9).These effects did not contradict our recent results obtained using plastic (polyethylene bottle) smoke preparations 17,18 and data of literature, where antioxidant properties, in particular the ability to act as ROS scavenger were shown for carbon dots and plastic organics [35][36][37][38][39] .ROS are very important for the regulation of neurotransmission (especially GABA-ergic one) and synaptic plasticity 40 , an inhibition of kainateinduced ROS production by MPs may result in the disruption of receptor-mediated signaling pathways, thereby indirectly influencing neuronal activity.It can be speculated that MP-induced changes in ROS production may contribute to the alterations in the extracellular levels of [ 3 H]GABA (and to less extent L-[ 14 C] glutamate) in nerve terminal preparations (Table 2).
MP-induced changes in the membrane potential of nerve terminals and isolated neurons (Fig. 6) were in line with our previous experiments on wood smoke-induced changes in the membrane potential of nerve terminals and cell viability in culture (gut COLO 205 cells) 17,18,41 .
The question rose from the obtained results regarding a mode of neuroactive action of MPs in nerve terminals.Our recent study concerning membrane active capability of modelling carbon dots synthesized from β-alanine demonstrated that they increased transmembrane current of cations in the suspended bilayer lipid membrane (BLM) by inducing stable potential-dependent cation-selective pores 42 .This in turn was coincided with impaired synaptic neurotransmission, in particular the increased extracellular level of excitatory neurotransmitters L-[ 14 C] glutamate, D-[ 2,33 H] aspartate, and the inhibitory ones [ 3 H] GABA, [ 3 H] glycine in the nerve terminals.Carbon-containing nanoparticles synthesised by combustion of organics significantly dysregulated presynaptic processes 43,44 .
Therefore, it can be expected that carbon nanoparticle component of MPs directly (moving through the plasma membrane to the cytosol of nerve terminals) or/and indirectly (through an interaction with the plasma membrane of nerve terminals, disturbance of its integrity and membrane-associated pathways) can influence functioning of intracellular organelles, such as mitochondria and synaptic vesicles.Indeed, the experiments using JC-1, a fluorescent cationic carbocyanine dye accumulated by mitochondria, showed that the mitochondrial potential was significantly (appx twice) dissipated by MPs (Fig. 7).Also, the measurements of synaptic vesicle acidification using a pH-sensitive fluorescent probe acridine orange revealed a dissipation of the proton gradient of synaptic vesicles in response to the application of MPs (Fig. 8).The principal difference between these two intracellular organelles regarding their potential interaction with MP-derived carbon nanoparticle component was the following: MP-induced pores formed by the nanoparticle component in the plasma membrane of the nerve terminals can be transferred to synaptic vesicles during permanent synaptic vesicle recycling, in contrast to mitochondria, where the nanoparticles should penetrate the plasma membrane to reach mitochondria and interact with the mitochondrial membrane.Also, it is not excluded that besides carbon nanoparticle components, other smoke compounds can also contribute to mitochondria response.In perspective, our further efforts will be focused on analysis of changes in mitochondria functioning in response to application of smoke preparations from different types of organics.Also, further experiments using in vivo animal models, inhalation approaches and new technique [45][46][47] are necessary to uncover detailed mechanisms of MPs action.
It is clear from our results that the neurotoxic effects of MPs were almost similar to the main neuroactive characteristics of smoke preparations synthesized from polyethylene bottles 17,18 .Therefore, accidental or intentional utilization of medical facemask waste by combustion can contribute to plastic-related air pollution of the environment.It has been suggested that the central nervous system is not substantially protected against evolutionary new challenges such as exposure to anthropogenic carbon-containing air pollution PM 32 , moreover an association of air pollution by PM and expansion of neurological/neurodegenerative disorders/disease was already proven 4,48 .Particles, especially nano-sized ones, can be deposited in humans in the nasal region, and move along the olfactory nerve axons straight to the brain, thereby circumstancing the blood brain barrier 49 .Our data can be useful in complex environment monitoring as potential essential variables for air quality estimation 50 .
Besides air pollution, PM released during facemask combustion can also contribute to plastic-related pollution of water resources 51 .MPs contained nano-and micro-particles (Fig. 1), and in situ these particles in combination with incompletely combusted ones can be suspended in water that occurs during rains, floods, fire control and extinguishing.Carbon-containing particles can become water soluble, and contaminate water resources, sea and ocean 52 .The plastic-derived particles can interact with fish and murine organisms, and then after feeding they can www.nature.com/scientificreports/accumulate in humans causing health threats.Despite utilization problems, plastic is currently used to produce many products because of its low cost, light weight, and high durability and is emerging as the dominant material causing marine pollution 51,[53][54][55] .It should be noted that PM can be neurotoxic itself and also due to adsorption of neurotoxic environmental components, in particular heavy metals 45,47 .

Collection of smoke aerosol from combustion of disposable medical facemask under laboratory conditions
Disposable medical facemasks used in this study were composed of polypropylene fibers 16 .Smoke from combustion of the facemasks was collected in the laboratory conditions during the entire flaming, mixed combustion phase, when the flaming and smoldering phases were present simultaneously, and smoldering phase.In particular, the facemasks (60 g) were combusted; smoke emissions were collected using a specially designed vacuum installation and bubbled through 48 ml of water.Water-suspended MPs enriched with the most neuroactive nano-sized PM 32 were obtained by filtration through glass microfiber filters with a pore diameter of 1.0 µm 17 .Obtained MPs were dried to measure the concentration of particulate smoke components.A concentration of dry matter in MPs was 700 µg/ml.

Physical, optical, fluorescent and surface properties of MPs
Dynamic light scattering Particle size in MPs was examined using dynamic light scattering with a laser correlation spectrometer Zetasizer Nano ZS, Malvern Instruments (UK) equipped with He-Ne laser (P max = 4 mW, 633 nm).
Zeta potential Z-potential of MP nanoparticles was determined using laser Doppler electrophoresis technique that was inbuilt in the Zetasizer Nano ZS, Malvern Instruments (UK).Measuring procedures were carried out in capillary cell DTS1070 at such parameters: scattered angle 17°; refractive index (RI) = 1.450, like as for carbon nanoparticles; dispersant -water (T = 25 °C, viscosity 0.8872 cP, RI = 1.330);Henry's function (ƒ(K α )) = 1.5 (took according Smoluchowski approximation for aqueous solutions); number of measurements − 5 (each measurement includes 50 runs for 10 s).

Optical and fluorescent properties
Absorption characteristics of MP colloid were investigated using UV-Vis spectrophotometer Cary Eclipse Varian 50 Scan with wavelength range limits from 190 to 1100 nm.Measurements were conducted in Hellma Analytics Quartz SUPRASIL® High Precision Cell (10 × 10 mm light path).Fluorescent properties of samples were determined using Agilent® Cary Eclipse spectrofluorimeter, equipped with a 75 kW Xenon flash lamp with Δpulse = 2 μs, two Czerny-Turner type monochromators and an 800 V PM detector.The lamp frequency is set to 100 Hz by default.

Raman and FTIR spectroscopy
The Raman spectra of MPs were monitored using a custom-made Raman instrument pre-verified, and equipped with a 40 × objective microscope.A Verdi G laser, Coherent Inc. with a wavelength of 532 nm was applied to excite MPs, the laser power on the sample was 30 mW, and the scattered light of samples was collected by the lens and filtered using two filters (RazorEdge 0° Longpass filter, Semrock).Raman scattered light was focused on a monochromator inlet slit (IsoPlane 320, Princeton Instruments) set at 30 μm to deflect unfocused light, and have a high spectral resolution.The monochromator was equipped with a diffraction grating of 600 lines/mm, and calculated spectral resolution was appx 2 cm −1 .A CCD (PyLoN: 400BR-eXcelon CCD, Princeton Instruments), cryogenically cooled at − 120 °C was used as a detector [18][19][20][21][22][23][24][25][26] .
MPs were analysed using FTIR spectrometer INVENIO-R (Bruker, Germany).The samples for the Raman scattering spectra, and infrared absorption were made by applying a drop of MP solution to a CaF 2 substrate 18 .

Ethical statement in animal experiments
Wistar rats, males, body weight of appx 100-120 g, were kept in a temperature-controlled room at 22-23 °C using the institute`s animal facilities, where the rats were provided ad libitum with water and dry food.Experiments involving animals were conducted in accordance with the Guidelines of the European Community (2010/63/EU), the ARRIVE guidelines for reporting experiments involving animals 56,57

Isolation of the rat cortex nerve terminals (synaptosomes)
The synaptosomal preparations were obtained using differential and Ficoll-400 density gradient centrifugation of rat brain homogenates according to the method described by Cotman with minor modifications 58 .The protein concentrations were recorded according to Larson 59 .MP aliquots were added to the synaptosomal suspensions and incubated for 10 min.

Isolation of neurons from the rat cortex
The rat cortex was dissected and connective tissue was removed.One cortex was used to obtain one preparation of neurons.The tissue was incubated in HEPES-buffered saline (10 mM HEPES, 150 mM NaCl, pH 7.4), then tissue was transferred to polystyrene tubes and incubated with protease from Streptomyces griseus type XIV using a shaking water bath at 30 °C.After inactivation of protease and washing procedure, the neurons were re-suspended using the siliconized 9-in Pasteur pipette 60 .

The transporter-mediated uptake of L-[ 14 C]glutamate by the nerve terminals
The initial rate of L-[ 14 C]glutamate uptake and its accumulation by synaptosomes was measured as follows.Synaptosome suspensions (125 μl of suspension, 0.4 mg of protein/ml) were pre-incubated with MP aliquots at 37 °C for 10 min.The uptake was initiated by the application of L-glutamate and L-[ 14 C]glutamate (10 µM and 450 nM, 0.167 µCi/ml, respectively).The synaptosomes were further incubated at 37 °C for 1 min (the initial rate) and 10 min (the accumulation), and then sedimented using a microcentrifuge (20 s at 10,000×g).Nonspecific binding of L-[ 14 C]glutamate was assessed in cooled samples after addition of radioactivity.L-[ 14 C]glutamate uptake was assessed as a decrease in radioactivity in the aliquots of supernatants (100 μl) and an increase in radioactivity in SDS-treated pellets 61 .L-[ 14 C]glutamate uptake was calculated with Sigma-Fluor® High Performance LSC Cocktail (1.5 ml) using liquid scintillation counter Hidex 600SL (Finland).L-[ 14 C]glutamate uptake data were collected in triplicate from several (n) experiments performed with different synaptosome preparations.

The transporter-mediated uptake of [ 3 H]GABA by the nerve terminals
The synaptosomes were diluted by the standard salt solution containing aminooxyacetic acid (100 μM), the protein concentration was 200 μg/ml.The synaptosome preparations were pre-incubated at 37 °C for 10 min, MP aliquots were added to the synaptosome suspensions, and the samples were further incubated for 10 min.
To determine the initial rate of [ 3 H]GABA uptake, the process was completed in 1 min by filtering aliquots through Whatman GF/C filters.After washing, the filters were dried, and then were suspended in the organic counting scintillant and counted using liquid scintillation counter Hidex 600SL (Finland).Nonspecific binding of [ 3 H]GABA was evaluated in cooled samples filtered immediately after the addition of radioactivity 62 .[ 3 H] GABA uptake data were collected in triplicate from several (n) experiments performed with different synaptosome preparations.

The exocytotic release and the extracellular level of L-[ 14 C]glutamate in the nerve terminal preparations
The synaptosome preparations (2 mg of protein/ml) after pre-incubation at 37 °C for 10 min were loaded with L-[ 14 C]glutamate (2.81 μM, 1 μCi/ml) at 37 °C for 10 min.After loading procedure, the synaptosome suspensions were washed with 10 volumes of ice-cold standard salt solution; the pellets were re-suspended to reach a final concentration of 1 mg of protein/ml.Synaptosome suspensions (125 μl; 0.5 mg of protein/ml) were pre-incubated at 37 °C for 10 min, then MP aliquots were added and incubated with synaptosomes for 10 min, and then sedimented using a microcentrifuge (20 s at 10,000 g).The extracellular level of L-[

The exocytotic and tonic release of [ 3 H]GABA and the extracellular level of [ 3 H]GABA in the nerve terminal preparations
The synaptosome preparations (2 mg of protein/ml) were pre-incubated at 37 °C for 10 min and loaded with [ 3 H]GABA (50 nM, 4.7 μCi/ml) for 10 min.GABA transaminase inhibitor aminooxyacetic acid (100 μM) was used during [ 3 H]GABA loading and release experiments to minimize the formation of GABA metabolites.After loading, the synaptosome suspensions were washed with 10 volumes of ice-cold standard salt solution, the pellets were re-suspended to have protein concentration of 1 mg of protein/ml.Synaptosome suspensions (120 μl) were pre-incubated at 37 °C for 10 min, then the aliquots of MPs were added and incubated for 10 min, and sedimented using a microcentrifuge (20 s at 10,000 g).The extracellular level of [

The membrane potential of the nerve terminals and neurons
The membrane potential of the nerve terminals and neurons was monitored using the fluorescent potentiometric dye rhodamine 6G (0.5 µM) based on its potential-mediated binding to the membranes 63 The synaptosome or neuron suspensions (0.2 mg of protein/ml) were pre-incubated at 37 °C for 10 min, and the measurements were carried out using a thermostated cuvette with continuous stirring.The synaptosome preparations and suspensions of neurons were equilibrated with the probe, and after that MP aliquots were added.The ratio (F) as an index of the membrane potential was calculated according to equation F = F t /F 0, where F 0 and F t were the fluorescence intensities of rhodamine 6G in the absence and presence of the synaptosomes and neurons, respectively.F 0 was estimated by the extrapolation of the exponential decay function to t = 0.The fluorescence measurements were carried out using a fluorescence spectrofluorometer QuantaMaster™ 40 (PTI, Inc., Canada) and a Hitachi 650-10S spectrofluorimeter at 528 nm (excitation) and 551 nm (emission) wavelengths.

The mitochondrial membrane potential of the nerve terminals
The cationic membrane-permeable dye JC-1, Mitochondrial Membrane Potential Assay Kit, was used to evaluate changes in the mitochondrial membrane potential of the nerve terminals.JC-1 showed potential-dependent accumulation by mitochondria, as evidenced by green fluorescence emission at (~ 529 nm) for the monomeric form of the probe, which was red-shifted (~ 590 nm) with concentration-dependent formation of red fluorescence J-aggregates 64 .The suspension of synaptosomes was incubated in a cuvette with a magnetic stirrer, the final protein concentration of which was 0.15 mg/ml, then JC-1 was added up to a final concentration of 2 µM, incubated in the dark at 37 °C for 10 min, and then the spectrum of the probe fluorescence was recorded at an excitation wavelength of 485 nm and an emission wavelength from 510 to 610 nm.Then, MP aliquots were added to the cuvette and the kinetics of probe fluorescence was monitored for 30 min at an excitation wavelength of 485 nm and an emission wavelength of 590 nm.Then, the MP-changed spectrum of the probe fluorescence was recorded at an excitation wavelength of 485 nm and an emission wavelength from 510 to 610 nm.For quantitative analysis, the ratio of the fluorescence intensity at 590 versus 530 nm was used, which allows to ignore the difference in the probe loading, the number of mitochondria and the probe fluorescence quenching by MPs.The fluorescence measurements were performed on a Hitachi 650-10S spectrofluorimeter.

Synaptic vesicle acidification in the nerve terminals
A pH-sensitive fluorescent dye, acridine orange, was selectively accumulated by the acid compartments of the nerve terminals, i.e., synaptic vesicles 65 .The changes in the dye fluorescence were measured using spectrofluorometer PTI QuantaMaster40 and Hitachi 650-10S at excitation and emission wavelengths of 490 and 530 nm, respectively.The reaction was started by the application of acridine orange (a final concentration of 5 μM) to synaptosome suspensions (a final concentration of 0.2 mg of protein/ml) pre-incubated in a stirred thermostatted cuvette at 37 °C for 10 min.Fluorescence (F) was calculated according to: F = F t /F 0 , where F 0 and F t were fluorescence intensities of the dye in the absence and presence of the nerve terminals, respectively.F 0 was calculated by extrapolation of exponential decay function to t = 0.

ROS in the nerve terminals
A cell-permeable non-fluorescent probe, 2′,7′-dichlorodihydro-fluoresceindiacetate (H2-DCFDA) was applied to monitor ROS production in the nerve terminals 17,18 .The probe became highly fluorescent upon oxidation after the intracellular de-esterification.The synaptosome suspensions (a final concentration of 0.2 mg of protein/ml) were incubated with H2-DCFDA (a final concentration of 5 μM) for 5 min in a stirred thermostated cuvette at 37 °C, and then the kinetic measurements were carried out.2′,7′-dichlorofluorescein (DCF) fluorescence changes were documented at excitation and emission wavelengths of 502 and 525 nm, respectively, (slit bands were 2 nm each) using a fluorescence spectrofluorometer QuantaMaster™ 40 (PTI, Inc., Canada).

Inclusion and exclusion criteria
The "n" value is present in the Figures and Tables for each experimental group of rats, and there were no exclusions among the experimental groups.The quality of synaptosomal isolation procedure from rat brains and synaptosome viability were controlled using specific criteria, i.e. the extracellular synaptosomal level of L-[ 14 C]glutamate, that characterized dynamic glutamate turnover across the plasma membrane of the nerve terminals 30,31 .
No data points were excluded from analyses because of their biologically implausible values.

Statistical analysis
The results were expressed as the mean ± S.E.M. of n independent experiments.Experimental data on the effects of control vs. MPs were analysed using one-way ANOVA.The accepted level of significance was set at p < 0.05.

Figure 1 .Figure 2 .
Figure 1.Dynamic light scattering diagrams of MPs.(a) Size distribution of MPs by intensity.(b) Size distribution of MPs by number.The graphs represent cumulative data analysis of five consequent measurements, each for 2 min.(c) Graph of Z-potential distribution between 5 measurements, pH 3.75, and each measurement included 50 runs for 10 s.Red line-first measurement, green-second, blue, black and pink-next following, correspondingly.

Figure 3 .
Figure 3. UV-VIS absorption spectrum of MPs in water.

Figure 4 .
Figure 4.The time course of the transporter-mediated uptake of L-[ 14 C]glutamate (a) and [ 3 H]GABA (b) by the nerve terminals in the presence of MPs at concentrations of 10, 50 and 200 μg/ml.Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001 as compared to the control; n = 15.

Figure 6 .
Figure 6.Fluorescence experiments: (a) The effects of MPs at concentrations of 10, 50 and 200 μg/ml on the membrane potential of the nerve terminals.(b) The effects of MPs at concentrations of 10, 50 and 200 μg/ml on the membrane potential of isolated cortex neurons.Synaptosome or neuron suspensions were equilibrated with rhodamine 6G (0.5 μM); when the steady level of the fluorescence was reached, MPs (marked by arrow) were added to synaptosome or neuron suspension.The trace represents 15 experiments performed with different preparations.

Figure 7 .
Figure 7.The mitochondrial membrane potential of the nerve terminals in the presence of MPs measured using the cationic membrane-permeable dye JC-1.(a) The time course of changes in JC-1 fluorescence.The dye (a final concentration of 2 µM) was added to the synaptosome suspension (a final protein concentration of 0.15 mg/ml), incubated for 10 min in the dark at a temperature of 37 °C.Then, MP aliquots were added to the cuvette and the kinetics of probe fluorescence was monitored for 30 min at an excitation wavelength of 485 nm and an emission wavelength of 590 nm.(b) JC-1 fluorescence spectra in the control and in the presence of MPs at a concentration of 50 μg/ml at 30 min time point recorded at an excitation wavelength of 485 nm and an emission wavelength from 510 to 610 nm.The trace represents 15 experiments performed with different preparations.(a) **p < 0.01; ***p < 0.001; as compared to the 0 min; n = 15.

Figure 8 .
Figure 8. Synaptic vesicle acidification in the presence of MPs at concentrations of 10, 50, 200 μg/ml.The synaptosome suspension was equilibrated with 5 µM acridine orange, and when the steady level of the dye fluorescence had been reached, MP aliquots (marked by arrow) were applied.The trace represents 15 experiments performed with different preparations.

Table 2 .
The extracellular level of L-[ 14 C]glutamate and [ 3 H]GABA in the nerve terminal preparations in the presence of MPs at concentrations of 10, 50 and 200 μg/ml.***p < 0.001; n.s., no significant differences as compared to the appropriate control.
The extracellular level of L-[

Table 3 .
Tonic release of [ 3 H]GABA from the nerve terminals in the presence of MPs at concentrations of 10, 50 and 200 μg/ml.
and local laws/policies.The protocol was approved by the Animal Care and Use Committee of the Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, the Protocol #1 from 28/01/2021.To follow a reduced animal number strategy, synaptosomal preparations were shared between radiolabeled and fluorescence experiments.The total number of rats used in this study was 15, among them 15 rats were used for L-[ 14 C]glutamate and [ 3 H]GABA transportation analysis; 15 shared rats − fluorescence measurements.
63C]glutamate in the nerve terminal preparations was measured at 6 min time point.The exocytotic release of L-[ 14 C]glutamate stimulated by membrane depolarization with 35 mM KCl, was measured at 6 min time point and calculated by subtraction of the release values in Ca 2+ -containing medium and Ca 2+ -free one.The extracellular level of L-[ 14 C]glutamate and its release were recorded in the aliquots of supernatants (100 μl) and pellets using liquid scintillation counter Hidex 600SL (Finland) and Sigma-Fluor® High Performance LSC Cocktail (1.5 ml), and the values were expressed as the percentage of total accumulated synaptosome label63.L-[ 14 C]glutamate release data were collected in triplicate from several (n) experiments performed with different synaptosome preparations.
63d measured in the presence of NO-711(30 μM)in Ca 2+ -containing medium at 6 min time point.[ 3]GABA radioactivity was measured in the aliquots of supernatants (90 µl) using liquid scintillation counter Hidex 600SL (Finland) and Sigma-Fluor® High Performance LSC Cocktail (1.5 ml), and the values were expressed as the percentage of total accumulated synaptosome [ 3 H]GABA63.[ 3 H]GABA release data were collected in triplicate from several (n) experiments performed with different synaptosome preparations.