Water-Soluble, Alanine-Modified Fullerene C₆₀ Promotes the Proliferation and Neuronal Differentiation of Neural Stem Cells

Abstract

As carbon-based nanomaterials, water-soluble C₆₀ derivatives have potential applications in various fields of biomedicine. In this study, a water-soluble fullerene C₆₀ derivative bearing alanine residues (Ala-C₆₀) was synthesized. The effects of Ala-C₆₀ on neural stem cells (NSCs) as seed cells were explored. Ala-C₆₀ can promote the proliferation of NSCs, induce NSCs to differentiate into neurons, and inhibit the migration of NSCs. Most importantly, the Ala-C₆₀ can significantly increase the cell viability of NSCs treated with hydrogen peroxide (H₂O₂). The glutathioneperoxidase (GSH-Px) and superoxide dismutase (SOD) activities and glutathione (GSH) content increased significantly in NSCs treated even by 20 μM Ala-C₆₀. These findings strongly indicate that Ala-C₆₀ has high potential to be applied as a scaffold with NSCs for regeneration in nerve tissue engineering for diseases related to the nervous system.

1. Introduction

Neural stem cells (NSCs) have the potential of self-renewal and multipotent differentiation, which can differentiate into neurons, astrocytes, and oligodendrocytes in the nervous system [1,2]. Because NSCs offer a potential source for cell/tissue replacement therapy, they are ideal candidates as seed cells for neural tissue engineering and regenerative medicine.

Many studies have suggested that the physical and chemical properties of scaffold materials, such as inorganic nanomaterials, have a critical effect on the self-renewal and neuronal differentiation of NSCs in neural tissue engineering [3]. For example, mesoporous silica nanoparticles have been found to be capable of loading neural growth factors to promote neural-cell and neurite growth, even to control the fate of NSCs [4] and help imaging of NSCs distribution in glioma xenografts [5]. Reduced graphene oxide (rGO)/TiO₂ heterojunction film can be used as a biocompatible flash photo stimulator for effective differentiation of hNSCs into neurons [6]. A nanostructured rGO microfiber was found to support NSCs growth and regulate NSCs differentiation into Tuj1-positive neurons while inhibiting the glial differentiation [7]. Graphene substrates were shown to promote the differentiation of human neural stem cells into neurons [8]. A dense TiO₂ ceramic with a flat surface can regulate NSCs into Tuj1-positive neural differentiation, while nanoporous TiO₂ ceramics can induce the glial differentiation of NSCs. These data proved that localized and committed differentiation can be achieved in NSCs culture system by integrating materials with different topographies [9]. Carbon nanotube (CNTs) promoted neurite elongation and the differentiation of NSCs into mature neuronal cells [10].

Among the carbon-based nanomaterials, fullerene C₆₀ (refers to C₆₀ hereafter), with a stable cage-like structure resistant to the actions of both acids and bases, is a promising material in various fields of biomedicine [11,12,13,14]. However, C₆₀ is a fully hydrophobic molecule and has poor solubility in water, which greatly limits its biomedical functions. To solve this problem, numerous preparation techniques have been developed, which resulted in a wide range of C₆₀ derivatives. Through extensive modification on the surface, water-dispersible C₆₀ derivatives could be obtained, opening the door for their utilization in biomedical fields including anticancer/antimicrobial therapy, controlled drug delivery, photodynamic therapy, and cytoprotection [15,16]. Specifically, in the nervous system, the hydroxylated C₆₀, i.e., fullerenols, have shown spectacular antioxidant activity to reduce apoptosis in cortical neurons [17]. Hexa (sulfobutyl) C₆₀ derivative and trimesic acid (TMA) C₆₀ derivative also demonstrated their ability to trap free radicals in the treatment of neurodegenerative diseases [18,19]. Fullerenols and malonic-acid-modified C₆₀ can reduce neuronal degeneration related to reactive oxygen species (ROS) by reacting with hydroxyl and superoxide free radicals [20]. Many studies showed the neuroprotective effect of C₆₀ derivatives as radical scavengers in in vitro and in vivo stroke models [21,22,23,24]. C₆₀ derivatives were found to affect the proliferation and differentiation of NSCs. Aligned C₆₀ nanowhiskers (NWs) can orient NSCs and have a high capacity to differentiate into mature neurons. The aligned C₆₀ NWs can serve as a functional scaffold for neural tissue engineering [25]. C₆₀ and CNTs were found to enhance the proliferation and differentiation of NSCs [26]. Water-soluble C₆₀ derivatives with different types of linkages between the C₆₀ cage and the solubilizing groups could exert different effects on neural stem cells and glioblastoma cells. The compound bearing residues of phenylbutiryc acids significantly increased the proliferation/survival of NSCs and induced neural repair, while the compound with phenylalanine appendages significantly inhibited the glioblastoma growth [27]. For neural tissue engineering, new water-soluble C₆₀ derivatives should be designed, and their effects on NSCs need to be investigated deeply.

In this study, we successfully assembled a water-soluble C₆₀ derivative modified with alanine (refers to Ala-C₆₀ hereafter). NSCs were used as seed cells to determine whether the Ala-C₆₀ can affect the proliferation, migration, and differentiation of NSCs. The results demonstrate that the Ala-C₆₀ not only promotes proliferation but also enhances neuronal differentiation under neural differentiation conditions for seven, while inhibiting the migration of NSCs. Most importantly, the Ala-C₆₀ can significantly increase the cell viability of NSCs treated with H₂O₂. GSH-Px and SOD activities and GSH content increased significantly in NSCs treated even by 20 μM Ala-C₆₀. This result proves the anti-oxidative stress effect of the Ala-C₆₀. This finding provides the possibility to utilize the Ala-C₆₀ as a scaffold with NSCs in neural tissue engineering.

2. Results

2.1. Preparation and Structure of Ala-C₆₀

Ala-C₆₀ was synthesized by referring to the literature [28] with modifications. The detailed preparation process can be found in the experimental section. Compared to the previous method, we employed o-xylene as a solvent for C₆₀ instead of toluene, which improved the yield of the final product due to the much higher concentration of C₆₀ in o-xylene than in toluene. The employment of dialysis during the purification process gives a complete removal of the inorganic salts and small organic molecules, making the final product highly pure.

The types of the organic functional groups on the surfaces of the Ala-C₆₀ were probed by FTIR measurement (Figure 1A). The vibration of N-H (νN-H) and C-N (νC-N) located at 3440, 1060 cm⁻¹ reveals the presence of amino groups on the molecules. The peaks at 2920 and 2849 cm⁻¹ indicate the presence of alkyl chains from residual tetrabutylammonium cations (TBA⁺). The peaks at 1574 and 1448 cm⁻¹ are from COO⁻, and the peak at 575 cm⁻¹ is the characteristic absorption of C₆₀. A three-step weight loss was confirmed (Figure 1B) from thermogravimetric analysis (TGA) performed under nitrogen. The first weight loss that occurred before 145 °C (11.43 wt%) is assigned to the loss of secondary bound water. The second weight loss before 238 °C (10.94 wt%) can be attributed to the dehydration of the hydroxy groups. The last one above 379 °C originated from the sublimation of C₆₀.

X-ray photoelectron spectroscopy (XPS) measurements were used to analyze the composition of Ala-C₆₀ (Figure 1C–F and Figure S1). In addition to C1s, O1s, and N1s, the presence of Na1s was also confirmed (Figure 1C and Figure S1). High-resolution C1s spectra (Figure 1D) showed peaks at 284.6, 286.5, and 287.7 eV, which can be assigned to C-N, C-C/C=C, and C-O, respectively, further confirming the connection of amino group and C₆₀. The atomic percentages of C, O, N, Na were determined to be 73.3%, 23.15%, 2.09%, and 2.46%, respectively. The weight ratio of C to H was calculated to be ~16 according to elemental analysis (

Table 1. The element analysis of Ala-C₆₀.

Weight (mg)	C (%)	H (%)	N (%)
3.0640	50.93	3.505	2.99

).

Based the above analyses, the average formula of Ala-C₆₀ was calculated to be C₆₀(NH-CH(CH₃)-COO⁻)₂O⁻₉H⁺₈Na⁺₂TBA⁺⋅9H₂O (Detailed calculations can be found in supporting information). Its structure is illustrated in Figure 2A.

2.2. Cytotoxic Effects of the Ala-C₆₀ on NSCs

The solubility of Ala-C₆₀ was investigated in detail in phosphate buffer solution (PBS), Dulbecco’s modified eagle medium (DMEM) and NSCs medium. As shown in Figure 2B, Ala-C₆₀ can dissolve in these three different mediums to give stable and transparent solutions. Next, the cytocompatibility of the scaffolds was determined using cultured primary NSCs. The primary NSCs derived from Sprague Dawley pregnant rat E13.5 brain. NSCs were cultured as monolayer adherent cells or as suspended neurospheres (Figure S2A,B). The cells expressed nestin, which is a common marker for NSCs (Figure S2C) [29]. NSCs were plated at a density of 1 × 10⁴ cells per well of a 96-well plate with NSCs medium for three days. Cell Counting Kit-8 (CCK) assay was performed after 48 h of incubation with Ala-C₆₀ protecting from light. The different concentrations of 10 to 320 μM of Ala-C₆₀ were applied in this assay. The concentration of Ala-C₆₀ with 160 and 320 μM significantly reduced the cell viability compared to the control group (160 μM: 63.11 ± 16.60%, p = 0.024 vs. CTR; 320 μM: 57.52 ± 9.34%, p = 0.006 vs. CTR). The cell viability showed no significant difference in groups treated with 10, 20, 40, 80 μM Ala-C₆₀ (p > 0.05, Figure 2C). This result suggests that Ala-C₆₀ had low toxicity on the survival of NSCs even at a relatively high concentration (≥160 μM). Therefore, the concentrations of 20, 40, and 80 μM were set for Ala-C₆₀ in the following experiments.

2.3. Promotion of the Proliferation and Neuronal Differentiation of NSCs by Ala-C₆₀

In order to test whether Ala-C₆₀ can affect the properties of NSCs, the proliferation and differentiation of NSCs were investigated. The detailed experimental scheme is shown in Figure 2D. First, for the proliferation assay, NSCs were cultured as neurospheres in proliferation medium for three days. Subsequently, NSCs were incubated with 20, 40, and 80 μM Ala-C₆₀ for 48 h in the dark. BrdU/nestin double immunofluorescence staining with antibodies against BrdU and nestin was used to label the proliferating NSCs. BrdU, a thymidine analog, can replace thymine (T) into the replicating DNA molecule during cell proliferation [30]. As shown in Figure 3A,B, the proportion of BrdU/nestin-positive cells in the group treated with Ala-C₆₀ was significantly higher than that in the control group (17.07 ± 2.77%), and the proportion increase depended on the increase in concentration of Ala-C₆₀. The proportions of BrdU/nestin-positive cells treated with 20, 40, and 80 μM Ala-C₆₀ were 29.87 ± 6.88%, 37.73 ± 3.51%, and 49.38 ± 5.29%, respectively. This result indicates that Ala-C₆₀ can promote the proliferation of NSCs.

Next, the NSCs as neurosphere suspension culture was induced to differentiate by the differentiation medium on the sixth day after plating. Neurospheres and differentiation medium were added to the 24-well plate after treatment with poly-L-lysine. Ala-C₆₀ was added into the medium after plating for 24 h. Interestingly, after one week of differentiation, the cells showed a spider web-like morphology compared to those in the control group (Figure 4A). On the seventh day after being treated with Ala-C₆₀, double immunofluorescence staining was performed. Microtubule association protein-2 (MAP2), an important component of the cytoskeleton of neurons, is also a specific marker of mature neurons [31]. Meanwhile, glial fibrillary acidic protein (GFAP) is a marker of astrocytes [32]. As shown in Figure 4B,C, with the dose increase of Ala-C₆₀, the proportion of MAP2-positive cells in the total number significantly increased, compared with the control group (5.32 ± 1.55%). The percentages of MAP2-positve cells treated with 20, 40, and 80 μM Ala-C₆₀ were 33.10 ± 5.18%, 56.30 ± 2.73%, and 79.39 ± 3.90%, respectively. Contrary to the increasing trend of MAP2-positive cells, the percentage of GFAP-positive cells was significantly reduced in the group treated with Ala-C₆₀. The percentages of GFAP-positive cells treated with 20, 40, and 80 μM Ala-C₆₀ were 29.75 ± 6.22%, 6.97 ± 0.61%, and 3.65 ± 1.00%, compared to the control group (50.63 ± 4.89%, Figure 4C). These results indicated that Ala-C₆₀ promoted neuronal differentiation while inhibiting glial differentiation.

2.4. Inhibition of the Migration of NSCs by Ala-C₆₀

In rodents, NSCs give rise to a large number of neuroblasts that migrate through the rostral migratory stream to the olfactory bulb, where they differentiate into interneurons. Conversely, in the human brain, a large number of neuroblasts migrate into the neocortex instead of the rostral migratory stream during the first few months of life [33]. Hence, migration is a vital process during neurogenesis.

To explore whether Ala-C₆₀ affected the migration of NSCs, the migration distance between NSCs progeny and neurospheres was measured during differentiation. After being treated with Ala-C₆₀ for 24 and 72 h, we measured the longest diameters of migrating differentiated cells to the neurospheres’ core in each group. The migration distance was 1104.39 ± 278.58 μm in the control group, while the migration distances in groups treated with different concentrations of Ala-C₆₀ were 711.36 ± 236.80 μm, 628.39 ± 321.08 μm, and 475.56 ± 193.04 μm, respectively (Figure 5A,B). The groups treated with Ala-C₆₀ had a significantly shorter migration distances compared to the control group. These data indicate that Ala-C₆₀ inhibited the migration of NSCs progeny. Moreover, the migration was inhibited more severely at a higher concentration of Ala-C₆₀.

2.5. Antioxidant Ability on NSCs of Ala-C₆₀ Induced by H₂O₂

H₂O₂ is the classical reagent to study oxidative stress. First, the capability of Ala-C₆₀ to scavenge the hydroxyl radical (OH·) produced by H₂O₂ was investigated by electron spin resonance (ESR) measurements. As shown in Figure 6A,B, the intensity of the peak decreased continuously with the increasing concentration of the Ala-C₆₀. The initial quenching of OH⁻ is pretty fast, with a quenching efficiency of up to 49.8% at a concentration as low as 4 μg·mL⁻¹ (∼5 ppm). It then slows down at relatively large concentrations of the Ala-C₆₀, which reaches ∼89% at 8.6 μg·mL⁻¹.

Next, we set up the oxidative stress model, using NSCs treated with H₂O₂ to explore the effect of Ala-C₆₀ on oxidative stress. In order to choose an optimal concentration of H₂O₂ and treatment time, we added various concentrations of H₂O₂ (100~2000 μM), respectively, to NSCs (Figure S3A–C). On the second day after neural stem cell seeding, H₂O₂ was added to the 96-well plate after poly-L-lysine adherence treatment, and the cell viability was measured at 24 h, 48 h, and 72 h. At 24 h, the concentration of H₂O₂ higher than 500 μM significantly reduced the cell viability (500 μM: 74.01 ± 5.91%; 1000 μM: 62.76 ± 0.87%; 2000 μM: 59.99 ± 1.05%; Figure S3A). At 48 h, the cell viability of NSCs treated with H₂O₂ (≥500 μM) fell drastically compared to the control group (500 μM: 64.56 ± 2.51%; 1000 μM: 51.67 ± 3.66%; 2000 μM: 41.02 ± 1.60%; Figure S3B). At 72 h, the cell viability of NSCs treated with H₂O₂ (≥500 μM) fell lowest compared to the control group (500 μM: 65.81 ± 7.86%; 1000 μM: 37.27 ± 5.49%; 2000 μM: 29.42 ± 0.62%; Figure S3C). Finally, we chose 500 μM H₂O₂ to treat NSCs for 24 h to establish an oxidative stress model. Thus, the concentration and time were determined in the following experiments.

In order to test the effect of Ala-C₆₀ on NSCs during oxidative stress, different concentrations of Ala-C₆₀ (20, 40, 80 μM) were added to NSCs medium treated with 500 μM H₂O₂ for 24 h. After 24 h, the cell viability was measured. In Figure 6C, cell viability of NSCs increased as the concentration of Ala-C₆₀ increased. Especially, treatment with 40 and 80 μM of Ala-C₆₀ significantly increased the cell viability of NSCs with oxidative stress (p = 0.023 and p = 0.004). This result suggests that Ala-C₆₀ can inhibit H₂O₂-induced cell damage.

SOD is an important component of the antioxidant enzyme system in organisms [34]. GSH-Px is an important enzyme that catalyzes the decomposition of hydrogen peroxide widely in the body [35]. GSH, which catalyzes H₂O₂ into H₂O, is the most important non-enzymatic antioxidant in the body and an important factor in measuring the body’s antioxidant capacity [36]. These indicators were used to evaluate the antioxidant capacity and repair ability of Ala-C₆₀ on the NSCs treated with H₂O₂. Figure 6D shows that the activity of the SOD enzyme was significantly reduced when NSCs treated with H₂O₂. However, treatment with 20 and 40 μM of Ala-C₆₀ for 3 h significantly increased the activity of the SOD enzyme (p = 0.03 and p = 0.007). Furthermore, oxidative stress can also cause a decrease in the content of GSH and the activity of GSH-Px. After treatment with 20 and 40 μM of Ala-C₆₀, the content of GSH almost recovered to the level of the control group, and the activity of GSH-Px was much higher than that of the control group (Figure 6E,F). These results prove that Ala-C₆₀ has the antioxidant ability and can also increase the activity of antioxidant enzymes and repair the damage of NSCs induced by H₂O₂.

3. Discussion

In the present study, we successfully synthesized a water-soluble fullerene C₆₀ derivative bearing alanine residues. A high concentration (≥160 μM) of Ala-C₆₀ had low toxicity on the survival of NSCs. The effects of Ala-C₆₀ on the property of NSCs were investigated. First, Ala-C₆₀ promoted the proliferation of NSCs, induced NSCs to differentiate into neurons, and inhibited the migration of NSCs. Most importantly, Ala-C₆₀ had a significant neuroprotective effect on the cell damage of NSCs induced by H₂O₂. The activity of antioxidant enzymes (GSH-Px and SOD) and the content of GSH increased significantly in NSCs treated with Ala-C₆₀. These findings strongly indicate that Ala-C₆₀ has high potential to be applied as a scaffold with NSCs for regeneration in nerve tissue engineering for diseases related to the nervous system.

There were active debates about the toxicity of C₆₀ and its derivatives in the past [37,38,39]. The toxicity of nine different types of water-soluble C₆₀ derivatives was investigated on the NSCs, where the IC₅₀ value was found to vary from 200 nM to 1.5 mM [27]. The different toxicity of C₆₀ derivatives can depend significantly on the chemical structure of the appended organic addends and on the solubility of the compounds in aqueous media. Our data show that Ala-C₆₀ had no significant effect on the cell viability of NSCs at a concentration lower than 160 μM. However, a relatively high concentration (160 μM and 320 μM) of Ala-C₆₀ reduced the cell viability. The low toxicity of Ala-C₆₀, combining its good solubility in various mediums, makes it suitable for study and application in biomedicine.

Two fundamental properties of NSCs are the ability to self-renew and to give rise to differentiated progeny. The self-renewal of NSCs can be performed by symmetric or asymmetric cell division. Symmetric cell divisions of NSCs generate two daughter cells sharing the same identity, which is also the same as that of the mother cell. In asymmetric self-renewing cell division, the two daughter cells generated from an NSC have different identities, but one of them shares the same identity as that of the mother cell [40]. In addition, NSCs can differentiate into lineage-specific cells, such as neurons, oligodendrocytes, and astrocytes [41]. As seed cells, it is assumed that NSCs are to provide a regenerative source for new neurons in neural tissue engineering and regenerative medicine. The self-renewal and differentiation of NSCs are affected by many intrinsic and extrinsic factors. Many C₆₀ derivatives have been shown to affect the proliferation and differentiation of NSCs [25,26,27]. Our data show that Ala-C₆₀ not only promotes proliferation but also enhances neuronal differentiation. Nevertheless, at present, how Ala-C₆₀ affects proliferation and differentiation remains an open question.

In neural tissue engineering, the differentiated cells derived from NSCs need to migrate to the destination. However, our data show Ala-C₆₀ has a negative effect on migration. These should be additional factors that can promote migration to help Ala-C₆₀ and NSCs in the application of neural tissue engineering. Like neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) has a chemoattractant effect in the migration of neuronal precursor cells along the rostral migratory stream [42]. Furthermore, for non-classical neurotrophic factors, mesencephalic astrocyte-derived neurotrophic factor (MANF), together with its homolog cerebral dopamine neurotrophic factor (CDNF), can promote the migration of neural progenitor cells in the stroke model [43,44].

It is essential for the safety of the biological system to balance the oxidation and antioxidant activity of the body. Massive free radical production by normal cellular metabolism, as well as abnormal reactions, cause cell damage. The structure of C₆₀ makes it capable of reacting with free radicals, such as superoxide, hydroxyl radicals, and hydrogen peroxide [45]. Two different fullerenes, C₆₀ and C₈₂, conjugated with three transition metals, were used as an antioxidant [46]. The antioxidant ability of C₆₀ has been widely used in cosmetics and skincare products as an antiageing agent [47]. Fullerene derivatives in Alzheimer’s disease had radical scavenging ability, ROS activation, and ability to interact with peptides [45]. Hydrogen peroxide originates from the enzymatic or spontaneous dismutation of superoxide anions, which are the byproducts of a wide and ubiquitous variety of oxidases [48]. H₂O₂ has widely been used to induce cellular oxidative stress in different cell lines. Previous studies have revealed that cultured NSCs are sensitive to the oxidative stress induced by H₂O₂ [48,49,50,51,52]. In this study, the activity of antioxidant enzymes (GSH-Px and SOD) and the content of GSH in NSCs reduced after treatment with H₂O₂. These findings were consistent with the previous studies [48,50]. Furthermore, our results show that Ala-C₆₀ has the antioxidant ability. Ala-C₆₀ may be a therapeutic candidate combined with NSCs for the treatment of neurological diseases mediated by oxidative stress.

In summary, we have synthesized a water-soluble fullerene C₆₀ derivative bearing amino acid (alanine) residues. With careful structural analysis, the average form of this C₆₀ derivative (Ala-C₆₀) was obtained. The effects of Ala-C₆₀ on the proliferation, differentiation, and migration of NSCs were explored in detail. Our findings reveal that Ala-C₆₀ can promote the proliferation of NSCs, induce NSCs to differentiate into neurons, and inhibit the differentiation of astrocytes. In addition, it can also significantly increase the antioxidant ability of NSCs after oxidative stress induced by H₂O₂. These findings strongly indicate that Ala-C₆₀ has high potential to be applied for regeneration in nerve tissue engineering for diseases related to the nervous system. Considering that the family of amino acids is rich and the synthetic methodology of water-soluble C₆₀ derivatives could be further optimized, we believe this type of C₆₀ derivatives could find a wide range of applications in biomedicine.

4. Materials and Methods

4.1. Chemicals and Reagents

C₆₀ (99.5%) was purchased from the Suzhou Dade Carbon Nanotechnology Co., Ltd. (Suzhou, China). Tetrabutyl ammoniumhydroxide (TBAH; 40 wt% in H₂O) was purchased from Sigma (St. Louis, MO, USA). Sodium hydroxide (NaOH, AR) and alanine (AR) were bought from the Sinopharm Chemical Reagent Co. (Shanghai, China). Other chemicals, including ethanol, o-xylene, and hydrochloric acid (HCl), were obtained from local suppliers with the highest purity. All the chemicals were used as received unless other stated.

4.2. Synthesis of Ala-C₆₀

The water-soluble C₆₀ derivative used in this study, named Ala-C_60, was synthesized following the procedures reported in the literature [28] with modifications. Briefly, a stock solution of C₆₀ in o-xylene was prepared with a concentration of 5 mg·mL⁻¹. Then, 0.884 g of alanine was added to 15 mL of NaOH aqueous solution (1 g·mL⁻¹). After alanine was totally dissolved, 45 mL of ethanol was added to obtain the stock solution of alanine. To a round-bottom flask containing 90 mL stock solution of C₆₀, the stock solution of alanine was added dropwise under stirring, followed by the addition of 10 drops of TBAH (40 wt%). The mixture was allowed to react in the dark at 25 °C until the upper organic phase became colorless. The dark brown bottom phase was separated with a separating funnel, and the upper phase was washed twice with water. Then, the aqueous phase was combined, and its pH value was adjusted to ~7.0 with HCl. After that, the solution was subjected to dialysis against water with a dialysis tube with a molecular cut-off of 100 Da. The conductivity of the exudate was monitored with a conductivity meter, and dialysis was stopped when the conductivity of the exudate was below 10 μS/cm. The solution was taken out and filtered to remove any precipitates. Finally, water was evaporated in an oven at 60 °C to obtain the target product in a crystallized form.

4.3. Isolation of NSCs and Cell Cultures

Following previous protocol [53], NSCs were obtained from E13.5 Sprague Dawley rats embryonic cortical neuroepithelium. The tissues were dissected and digested with 0.2% papain (Sigma, St. Louis, MO, USA) at 37 °C for 30 min. Cells were counted and plated in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% B₂₇ (Invitrogen, Carlsbad, CA, USA), 20 ng·mL⁻¹ recombinant human EGF, and 20 ng·mL⁻¹ recombinant human basic FGF (both purchased from Millipore, Billerica, MA, USA). NSCs were cultured as adherent cells or suspended neurospheres. For the adherent culture, plates were coated with poly-L-lysine before the cell plating. However, for suspended neurosphere culture, no coating was needed. NSCs were passaged approximately every seven days.

4.4. Cell Viability Assay

Cell Counting Kit-8 (CCK) assay purchased from Biosharp (Hefei, China) was used to analyze cell viability. The concentrations of Ala-C₆₀ were 10, 20, 40, 80, 160, 320 μM. Neural stem cells were plated as density of 1 × 10⁴ cells/well in 96-well plates for three days and then exposed to Ala-C₆₀ for 48 h. The original medium was discarded, and 100 μL fresh medium sample was added to each well, together with 10 μL of CCK-8 solution. Absorbance was measured at 450 nm by a microplate reader.

4.5. BrdU Incorporation

We investigated the effect of Ala-C₆₀ on the proliferative activity of NSCs by bromodeoxyuridine (BrdU, Abcam, Cambridge, MA, USA) incorporation. NSCs were plated onto poly-L-lysine-coated coverslips at a density of 1.5 × 10⁴/well in 24-well plates. After 72 h, Ala-C₆₀ with different concentrations (20, 40, and 80 μM) were added to the 24-well plate, respectively, followed by the addition of 5 μL BrdU to each well on the fifth day. Two hours later, the cells were fixed in 4% paraformaldehyde and processed for immunofluorescence staining.

In brief, the cells were incubated in 2 N hydrochloric acid for 30 min and 10% donkey serum (DS) for 30 min at room temperature. Cells were incubated overnight with sheep polyclonal anti-BrdU antibody (1:400; Abcam, Cambridge, MA, USA) and mouse monoclonal anti-nestin antibody (1:400; Millipore, Billerica, MA, USA) in TBS containing 0.4% Triton X-100 at 4 °C. After three washings with PBS, the cells were incubated by the secondary antibody solution containing donkey anti-mouse Alexa-Fluor 488 (1:500; Abcam, Cambridge, MA, USA) and donkey anti-sheep Alexa-Fluor 594 (1:500; Life Technologies, Carlsbad, CA, USA) in TBS-Triton supplemented with DS for 30 min at room temperature.

4.6. Differentiation Assay and Immunocytochemistry Staining

NSCs were allowed to differentiate for seven days in a differentiation medium supplemented with 1% fetal bovine serum (FBS) and without EGF and bFGF. To the medium, Ala-C₆₀ with varying concentrations were added. On the seventh day of differentiation, the cells were fixed by 4% paraformaldehyde (PFA) for 10 min at room temperature for immunostaining of neuronal and astrocyte markers. The cells were washed three times with PBS to remove PFA, and then incubated with 10% donkey serum for 30 min at room temperature. The cells were further incubated overnight at 4 °C with primary antibody neuronal marker, rabbit polyclonal anti-microtubule-associated protein 2 (MAP2; 1:200) (Servicebio, Wuhan, China), and glial marker, mouse monoclonal anti-glial fibrillary acidic protein (GFAP; 1:500) (Servicebio, Wuhan, China) in TBST (TBS + 0.4% Triton-X) supplemented with normal donkey serum. After three washings with PBS, the cells were incubated by the secondary antibody solution containing donkey anti-mouse Alexa-Fluor 488 and donkey anti-rabbit Alexa-Fluor 594 (1:500; Abcam, Cambridge, MA, USA) in TBS-Triton supplemented with DS for 30 min at room temperature in the dark. Thereafter, the cells were nuclearly stained by incubating in an anti-fluorescence quencher containing DAPI (1:1000) for 5 min. A fluorescent microscope (Olympus, Allentown, PA, USA) was utilized to capture representative pictures to obtain merging images using ImageJ software. The values of neurons and astrocytes were counted, and the mean of each was calculated as a percentage.

4.7. Measurement of SOD, GSH-Px Sctivity and GSH Content

Inoculate NSCs into a 6-well plate for suspension culture. NSCs were treated with H₂O₂ (Sigma, 3 wt% in H₂O, St. Louis, MO, USA) for 24 h to obtain cells damaged by oxidative stress. After treatment with Ala-C₆₀ for 3 h, the cells were collected. Cell lysates were prepared with PBS or cell lysis buffer and centrifuged at 4 °C, 12,000 rpm, 20 min. SOD activity, GSH-Px activity and GSH levels were measured separately using a commercially available assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The SOD activity and GSH-Px activity, and GSH levels of neural stem cells were calibrated by protein content, and the protein determination was measured by the BCA method (Solarbio, Beijing, China).

4.8. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8. Graphs for each parameter were plotted for the averages obtained, and the error bars represented the SEM of the values. All data presented are means or means ± standard deviations. According to a one-way ANOVA, the variance in different groups was statistically analyzed to evaluate its significance, and p < 0.05 were recognized as statistically significant.