Abstract
Parkinson’s disease (PD) is one of the most aggressive neurodegenerative diseases and characterized by the loss of dopamine-sensitive neurons in the substantia nigra region of the brain. There is no any definitive treatment to completely cure PD and existing treatments can only ease the symptoms of the disease. Boron nitride nanoparticles have been extensively studied in nano-biological studies and researches showed that it can be a promising candidate for PD treatment with its biologically active unique properties. In the present study, it was aimed to investigate ameliorative effects of hexagonal boron nitride nanoparticles (hBNs) against toxicity of 1-methyl-4-phenylpyridinium (MPP+) in experimental PD model. Experimental PD model was constituted by application of MPP+ to differentiated pluripotent human embryonal carcinoma cell (Ntera-2, NT-2) culture in wide range of concentrations (0.62 to 2 mM). Neuroprotective activity of hBNs against MPP+ toxicity was determined by cell viability assays including MTT and LDH release. Oxidative alterations by hBNs application in PD cell culture model were investigated using total antioxidant capacity (TAC) and total oxidant status (TOS) tests. The impacts of hBNs and MPP+ on nuclear integrity were analyzed by Hoechst 33258 fluorescent staining method. Acetylcholinesterase (AChE) enzyme activities were determined by a colorimetric assay towards to hBNs treatment. Cell death mechanisms caused by hBNs and MPP+ exposure was investigated by flow cytometry analysis. Experimental results showed that application of hBNs increased cell viability in PD model against MPP+ application. TAS and TOS analysis were determined that antioxidant capacity elevated after hBNs applications while oxidant levels were reduced. Furthermore, flow cytometric analysis executed that MPP+ induced apoptosis was prevented significantly (p < 0.05) after application with hBNs. In a conclusion, the obtained results indicated that hBNs have a huge potential against MPP+ toxicity and can be used in PD treatment as novel neuroprotective agent and drug delivery system.
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Introduction
Parkinson’s disease (PD) is defined as one of the most common degenerative neurological diseases with motor and non-motor symptoms. It is characterized pathologically by the loss of neurons in the substantia nigra (SN) region where is a basal ganglia structure located in the midbrain that plays an important role in the movement. The definitive diagnosis of the disease can be performed only in the histopathological examinations of the brain tissue after death (Jankovic and Kapadia 2001). Although the source of PD is still unclear, some studies show that it is caused by oxidative stress and mitochondrial dysfunction in neurons (Stefanis et al. 1997). To study PD in vitro or to produce a PD cell model, there is a common method to obtain dopaminergic neuronal loss that contains the use of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), formed after MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) metabolism. MPP+ is able to induce parkinsonism and transported to the mitochondria, where it inhibits complex I, interacts with mitochondrial respiration and this interaction increases the amount of superoxide anions (Singer et al. 1987). This formation damages energy production and leads to increased free radicals in the cell, eventually causing neuron death (Lee et al. 2011). As recent studies have described that MPP+ toxicity on dopaminergic neurons could result in apoptosis through oxidative stress and inflammation (Esteves et al. 2008; Subramaniam and Chesselet 2013). The human embryonal carcinoma (EC) stem cell line (NTera2) was investigated to show similar characteristics with human embryonic stem cells (hESCs) (Pleasure and Lee 1993). Also, differentiation of NTera2 cell line by administration with all-trans retinoic acid was analyzed to give rise to human mature neuron-like cells and this cell culture was shown to be useful in many applications for in vitro brain disease studies (Lee and Andrews 1986; Zeller and Strauss 1995). Therefore, various studies recommended utilizing differentiated NTera2 cell line as in vitro Parkinson’s disease model to understand the disease from different perspectives (Schwartz et al. 2005; Schlachetzki et al. 2013).
Boron is found in nature as boron salts and is one of the basic components used by plants and humans as microelements (Gerretsen and de Hoop 1954; Haas 2002). Boric acid (BA) occurs in nature as a salt compound of boron. Several studies were reported that boric acid has antioxidant (Herrero et al. 2005), anti-genotoxic (Turkez 2008), anti-carcinogenic (Turkez and Geyikoglu 2010), non-cytotoxic (Turkez et al. 2010) and metal chelating (Turkez et al. 2012; Üstündaǧ et al. 2014) properties. On the other hand, boron nitride (BN) consists of boron and nitrogen elements and it can occur in different forms (hexagonal, cubic, sheets etc.) at various temperatures and pressures but, under normal conditions, the most stable form of BN is the hexagonal form (Merlo et al. 2018). It was reported that boron nitride nanotubes did not show any toxic effects on the kidney cells and BN nanotubes were shown to have good biocompatibility properties (Chen et al. 2009). A recent in vivo study revealed that G-chitosan-coated BN nanoparticle has non-toxic effects on the marginal ear vein of male rabbits (Ciofani et al. 2012). Again, a recent investigation asserted that boron nitride nanoparticles showed no cytotoxic potential on cultured healthy human alveolar cells and suggested that these nanoparticles could be used in pharmacological and medical applications safely (Türkez et al. 2019a).
In this study, it was aimed to investigate the effects of well-characterized hexagonal boron nitride nanoparticles (hBNs) against MPP+-induced neurotoxicity on neuron-like differentiated NT-2 cell culture to be able to explore novel uses area of boron nanoparticles in biomedical purposes. Firstly, NT-2 cells were transformed into neuron-like cell structure after treatment with all-trans retinoic acid and then an experimental PD model was formed with a certain dose of MPP+ treatment. Subsequently, hBNs were administered at various doses to determine biocompatible concentrations on transformed NT-2 cells and cell viabilities were determined by various cytotoxicity assays. Oxidative alterations were assessed with total antioxidant status (TAS) and total oxidant status (TOS) tests. Acetylcholinesterase (AChE) enzyme activity was also analyzed. Finally, the effects of hBNs on MPP+ induced apoptosis was determined by flow cytometry analysis.
Materials and methods
Synthesis and characterization of hBNs
For the synthesis of hBNs, 5 g boric acid (H3BO3) and 16 g sodium azide (NaN3) was dissolved in 600 ml deionized water. The mixture was agitated in the magnetic stirrer for 30 min then; 4.6 ml of N2H4.H2O were added slowly. The sample was mixed in the magnetic stirrer for half an hour again. The mixture was placed in the autoclave in the presence of an inert gas. The autoclaved samples were incubated in the ash oven at 300 °C for 16 h. Then the mixture was washed with deionized water to purify and samples were dried under vacuum at 100 °C for 2–3 h.
Surface and size analysis of hBNs were performed by scanning electron microscopy (SEM) (Zeiss Sigma 300®). The primary characterization of hBNs was performed by X-ray diffraction (XRD) method. XRD measurement was carried out at the Eastern Anatolia Technology Center (DAYTAM, Atatürk University, Erzurum, Turkey) using the Empyrean® (PANalytical®, Germany) model XRD. Analyzes were executed in 0.1 steps between 2θ = 10–90° and X-rays with a wavelength of 1.5406 Å.
Cell culture and neuronal transformation
Human malignant pluripotent embryonic carcinoma Ntera-2/D1 (NT-2/D1) cells (ATCC® CRL-1973) were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (v/v) inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin antibiotics. Cells were incubated at 37 °C, 5% CO2 and 95% air atmosphere. When cells were reached %80 confluences in the flask, dead cells were removed from the culture via washing with phosphate buffer saline (PBS). Cultures were incubated with a new medium containing 5% FBS with 10 μM concentration of all-trans-retinoic acid (all-trans-RA) (Sigma Aldrich®). The next day, the medium was replaced with a new medium containing 10% FBS without RA. This cycle was repeated for 11 days and changes in cells were photographed under an invert microscope (Andrews 1984; Andrews et al. 1990). Cell cycle phase distributions were investigated by using flow cytometry cell cycle analysis to confirm differentiations.
To constitute experimental Parkinson’s disease model, 1-methyl-4-phenylpyridinium (MPP+ iodide) (Sigma Aldrich®), an active metabolite of dopaminergic neurotoxin MPTP, was applied into differentiated cells at various concentrations (0.62, 0.12, 0.25, 0.50, 1 and 2 mM) and cell viability tests were performed after 24 h. The IC50 concentration of MPP+ was determined due to the results of viability tests. The differentiated cells were introduced with MPP+ at IC50 concentration to form the experimental PD model. hBNs nanoparticles were dispersed in deionized water and applied into the cultures in a wide spectrum range (0.19, 0.39, 0.78, 1.56, 3.16, 6.25, 12.50, 25, 50 and 100 mg/L). The untreated differentiated NT-2 cells were used as negative control and 1% Triton X-100 was used as a positive control. Each experimental sample was analyzed as triple replicates.
MTT cell viability assay
After 24 h, 10% of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well of a 96-well plate and it’s incubated for 3 h at 37 °C, 5% CO2 and 95% air atmosphere. After 3 h, supernatant from wells was aspirated. Formazan crystals formed inside cells were dissolved with DMSO (Sigma-Aldrich®) and measurements were performed with a microplate reader at a wavelength of 570–630 nm (Türkez et al. 2019b).
LDH release assay
Commercially available LDH kits (Thermo Fisher Scientific Massachusetts, USA.) were used according to the manufacturer’s instructions to determine the level of LDH release in the cell cultures. At the end of 24 h, 50 μL of the supernatant on cells was transferred to a new 96-well plate, 50 μL of the reaction mixture was added and incubated for 30 min in the dark at room temperature. Finally, 50 μL of the stop solution was added to wells and it was measured at 490–680 nm wavelengths by a microplate reader (Türkez et al. 2019b).
Total antioxidant status and total oxidant status analysis
Total antioxidant status (TAS) and total oxidant status (TOS) levels were calculated according to the manufacturer’s instructions (Rel Assay Diagnostics®, Turkey) (Emsen et al. 2016, 2018a, b).
Nuclear integrity analysis with Hoechst 33258 staining
After 24 h of incubation, the supernatant was removed and cells were washed with PBS. To fix the cells, the cultures were incubated for 30 min at +4 °C in the presence of 4% paraformaldehyde. After fixation, the cells were washed again with PBS and incubated with Hoechst 33258 (Sigma-Aldrich®) fluorescent dye in the dark for 5 min to stain the cell nucleus. Finally, the cell nuclei were analyzed under a fluorescent microscope (Leica® DM IL LED) (Alak et al. 2018).
Measurement of AChE activity
AChE activities in the cultures were analyzed calorimetrically using the Acetylcholinesterase Assay Kit from Abcam® (Cambridge, MA, USA). According to the manufacturer’s instructions, 50 μL of supernatant was transferred to a new plate and 50 μL of the master mix was added and stored for 30 min at room temperature protected from light. Finally, each plate well was measured at 410 nm wavelength by a microplate reader (Marinelli et al. 2017).
Flow cytometric analysis
Cells were detached from the bottom of the cell culture plates by trypsinization at the end of the incubation for 24 h and collected by centrifugation. At the end of the centrifugation, the pellet was suspended in 500 μL of 1x binding buffer. Then, suspended cells were incubated with 5 μL of Annexin V-FITC dye and 5 μL of propidium iodide dye. After 5 min of incubation at room temperature in the dark, cells were analyzed by flow cytometry (CyFlow® Cube 6, Germany).
Statistical analysis
The numerical data obtained from experiments were analyzed with GraphPad Prism® version 7.0 statistical program. One-way ANOVA, Tukey and Dunnett tests were used for multiple comparison evaluations. Statistically significance level was accepted as % 95 (p < 0.05).
Results
Characterization of hBNs
Surface morphology and elementary analyzes of nanoparticles were performed by SEM and XRD techniques. Using different scales, hBNs were imaged under a SEM microscope (Fig. 1). Images showed that size and dispersion of the hBNs were homogeneous and particles have a size between 100 and 400 nm.
XRD analysis of hBN nanoparticles was performed using the spray pyrolysis method previously reported by Mooney and Radding (2003). XRD analysis showed that the highest peak 2θ = 26.76° corresponding to Müller index 002 plane (Fig. 2). Other peaks were observed at 2θ = 41.70°, 43.91°, 55.12° and 75.97°, respectively. The planar order of these peaks according to the Müller index is 100, 101, 004 and 110. Then the size of hBNs was calculated using the Debye-Scherrer formula (Patterson 1939).
D represents the size of the particle, λ is X-ray beam wavelength, β is the half of the maximum radian peak value (FWHM), θ represents the Bragg angle. D value of the highest peak in 002 plane was calculated as 220 nm.
Cell culture and neuronal transformation
NT-2 cell cultures were treated with all-trans RA for 11 days to differentiate into mature neuron-like cells (Esteves et al. 2015); Andrews et al. 1984; Kawasaki and Taira 2012) and cell transformations were investigated under a fluorescent microscope. After the application of RA, NT-2 cell shapes were elongated and cellular structures were developed like axon-dendrite interactions (Fig. 3). Further cell differentiation confirmations were performed by flow cytometry cell cycle analysis and according to results cell cycle tendencies were change from S phase to G1 phase (Table 1).
hBN NPs application increased cell viability in the experimental PD model
Primarily, in order to form an experimental PD model, differentiated NT-2 cells were exposed to various doses of MPP+ for 24 h. At the end of 24 h, MTT and LDH release tests were applied to differentiated NT-2 cells to determine the IC50 concentration of MPP+. According to the results of MTT and LDH release tests, the IC50 concentration of MPP+ was determined as 1 mM (Fig. 4).
In this study, to explore their cytotoxic damage potential, hBNs were applied to differentiated NT-2 cultures at various concentrations (0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50, 25, 50 and 100 mg/L). At the end of the 24-h cell viability in differentiated NT-2 cultures was calculated and presented in Fig. 5. After application with 100 mg/L of hBNs, cell viability was reduced (approximately %60) as compared to untreated cultures. However, applications with hBNs at concentrations lower than 6.25 mg/L did not lead any statistically significant (p > 0.05) alterations as compared to the control group.
To determine neuroprotective properties of hBNs against MPP+-induced neurotoxicity in the experimental PD model, hBNs were added into the well plates at different concentrations and cell viability was calculated after 24 h. After the application of 3.12 mg/L of hBNs, the observed cell viability rates were increased (13%) as compared to MPP+ applied group. Likewise, 6.25 mg/L of hBNs led to increases in cell viability by approximately 6%. In other groups, cell viability decreased in a dose-dependent manner (Fig. 6). The most effective concentration of hBNs was determined as 3.12 mg/L.
TAS levels were increased and TOS levels were decreased against hBN applications
In this study, the effects of hBNs on oxidative alterations of neuronal cultures, TAS and TOS levels were determined. MPP+ led to significant (p < 0.05) decreases in total antioxidant capacity and increases of the total oxidant level in differentiated NT-2 cells in comparison to untreated cultures. On the contrary, hBNs supported TAS levels without increasing the TOS levels. And, applications with hBNs led to statistically significant differences in differentiated NT-2 cells in the aspect of TAS and TOS values as compared with the positive control group (Table 2).
hBN NPs treatment protected chromosomal integrity
To examine the effects of hBNs on chromosomal structures of cultured cells, 3.12 mg/L of hBN was applied to the in vitro PD model for 24 h. Then, cells were stained with Hoechst 33258 and examined under a light microscope and the changes in nucleus structures in the cells were photographed. It was determined that chromosomes in cells were distorted and nuclear fragments were formed in cells of the in vitro PD model. On the other hand, chromosomal integrity was found to be similar to healthy cells after applications with hBNs in contrast to the positive control. In only hBNs applied group, no significant change was observed in the chromosomal structures as compared to the control group. In the experimental PD model with the application with hBNs, it was observed that the number of chromosomal distortions significantly decreased and the number of healthy cells significantly increased when compared to the MPP+ treated cultures (Fig. 7).
AChE activity slightly altered after hBN NPs exposure
AChE enzyme activity was analyzed after application with hBNs at a concentration of 3.12 mg/L for 24 h. The results of the analysis showed that AChE activity of MPP+ group was significantly decreased as compared to the untreated controls. On the other hand, there was no statistically significant difference in AChE activity between hBNs applied groups and the untreated group. AChE enzyme activity level slightly increased after application with hBNs in experimental PD model (Table 3).
hBN NPs ameliorated apoptosis in experimental PD model
After the application of hBNs the cell death mechanisms were analyzed by flow cytometry assay. Only the MPP+ application was found to induce apoptosis in the experimental PD model. However, the application of only-hBNs to differentiated NT-2 cells did not cause any change in the cell viability as compared to untreated controls. It was calculated that co-application hBNs with MPP+ increased the cell viability rates from %63.49 to %93.14 as compared to only treated with MPP+ (Fig. 8).
Discussion
On XRD patterns of hBNs, there are four peaks at the Bragg angle that correspond to (002), (100), (101), (004) and (110) planes of hBNs, respectively. These peaks seen in XRD results showed that hBNs have a hexagonal structure (Korsaks 2015; Jedrzejczak-Silicka et al. 2018). Cell transformation studies have been performed for many years. RA treatment was reported to lead embryonal carcinoma cells to neuronal transformation. Again, various studies have shown that cells were transformed into neuron-like morphology and left the cell cycle by RA-induction (Jones-Villeneuve et al. 1983; Marzinke et al. 2010; Mori et al. 2013). Also, it was shown that increased Vtr protein in the Neuro2a cells after RA treatment and this increase differentiated cell morphology from multipolar to bipolar structure (Shimizu et al. 2002; Sugahara et al. 2019). In parallel to these observations, in our study, NT-2 cells were transformed into a neuron-like cell structure with all-trans RA application. MPP+ which is a metabolic product of MPTP, has been discovered as a selective neurotoxin to the substantia nigra region of the brain and produces Parkinson-like toxicity (Parkinsonism) in cell cultures (Langston et al. 1984; Beal 2001; Janhom and Dharmasaroja 2015). Neurotoxin of MPP+ was generally used to generate the in vitro experimental PD model (Lee et al. 2011). In this study, the experimental PD model was constituted via using the IC50 dose of MPP+ compound and resulted in apoptotic death in differentiated NT-2 cell culture.
Recent studies showed that hBNs didn’t exhibit any toxic properties on human normal skin fibroblasts and human alveolar cells at a wide spectrum of concentrations (Kıvanç et al. 2018; Türkez et al. 2019a). Our results correlated with the literature that hBNs didn’t cause any toxicity at defined concentrations used in differentiated NT-2 cell culture with RA. Moreover, cell viability tests propounded that certain concentrations of hBNs were neuroprotective against MPP+-induced neurotoxicity. In previous studies, it was reported that reactive oxygen species (ROS) were increased in neurodegenerative diseases, especially in PD, and consequently resulted in neuronal loss in the substantia nigra region (Kim et al. 2015; Niedzielska et al. 2016). It was found that MPP+ used to form an in vitro model selectively inhibited the NADH CoQ reductase (complex I) of the mitochondrial electron transport chain (causing a dramatic reduction in ATP production) and thereby induced neuronal degeneration (Sanders and Greenamyren 2013). Furthermore, it was shown that MPP+ increased H2O2-related ROS species in neurons and led to apoptosis in cell cultures (Sheehan et al. 1997; Chung et al. 2001; Shimohama et al. 2003; Janhom and Dharmasaroja 2015). According to our results, hBNs applications were significantly increased antioxidant levels, ameliorated oxidative stress and prevented apoptotic cell death caused by MPP+ toxicity. Previous studies were investigated that MPP+ neurotoxin reduced the AChE enzyme activity in cells in a concentration-dependent manner as compared to the control group (Zang and Misra 1993). Present results supported these data that MPP+-induced neurotoxicity inhibited AChE enzyme activity in the experimental PD model. However, the application of hBNs wasn’t completely successful for ameliorating inhibitory effects of MPP+ on AChE activity. In this context, it can be concluded that treatment with hBNs might increase the level of antioxidants in the experimental PD model and reduce apoptosis-mediated cell death by MPP+ neurotoxicity.
Conclusions
In this study, neuroprotective properties of hBNs were demonstrated for the first time in the experimental PD model induced by MPP+. Cell viability tests were determined that hBNs do not exhibit neurotoxicity potentials. Total antioxidant capacity and total oxidant status analysis showed that hBNs led to increases of TAS levels and AChE activity and decreases of TOS levels in PD model as compared to only MPP+ applied group after 24 h. Finally, in the flow cytometry analysis, it was determined that the application of hBNs model significantly decreased the apoptotic cells in the experimental PD model. In the light of these findings; it can be suggested that hBNs can be used as an adjuvant or drug delivery system in the treatment of PD, since hBNs exhibit antioxidant and neuroprotective properties.
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Küçükdoğru, R., Türkez, H., Arslan, M.E. et al. Neuroprotective effects of boron nitride nanoparticles in the experimental Parkinson’s disease model against MPP+ induced apoptosis. Metab Brain Dis 35, 947–957 (2020). https://doi.org/10.1007/s11011-020-00559-6
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DOI: https://doi.org/10.1007/s11011-020-00559-6