Water-Soluble Fullerene Derivatives as Brain Medicine: Surface

6 Mar 2017 - Delivering drugs to the central nervous system (CNS) is a major challenge in treating CNS-related diseases. Nanoparticles that can cross ...
0 downloads 0 Views 4MB Size
Download PDF
Research Article www.acsami.org

Water-Soluble Fullerene Derivatives as Brain Medicine: Surface Chemistry Determines If They Are Neuroprotective and Antitumor Fu-Yu Hsieh,† A. V. Zhilenkov,§ I. I. Voronov,§ E. A. Khakina,§ D. V. Mischenko,§ Pavel A. Troshin,*,∥,§ and Shan-hui Hsu*,†,‡,⊥ †

Institute of Polymer Science and Engineering and ‡Research and Development Center for Medical Devices, National Taiwan University, Taipei 10617, Taiwan, R.O.C. § Institute for Problems of Chemical Physics of Russian Academy of Sciences, Semenov Prospect 1, Chernogolovka 142432, Russian Federation ∥ Skolkovo Institute of Science and Technology, Moscow 143005, Russian Federation ⊥ Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan 35053, Taiwan, R.O.C. S Supporting Information *

ABSTRACT: Delivering drugs to the central nervous system (CNS) is a major challenge in treating CNS-related diseases. Nanoparticles that can cross blood− brain barrier (BBB) are potential tools. In this study, water-soluble C60 fullerene derivatives with different types of linkages between the fullerene cage and the solubilizing addend were synthesized (compounds 1−3: C−C bonds, compounds 4−5: C−S bonds, compound 6: C−P bonds, and compounds 7−9: C−N bonds). Fullerene derivatives 1−6 were observed to induce neural stem cell (NSC) proliferation in vitro and rescue the function of injured CNS in zebrafish. Fullerene derivatives 7−9 were found to inhibit glioblastoma cell proliferation in vitro and reduce glioblastoma formation in zebrafish. These effects were correlated with the cell metabolic changes. Particularly, compound 3 bearing residues of phenylbutiryc acids significantly promoted NSC proliferation and neural repair without causing tumor growth. Meanwhile, compound 7 with phenylalanine appendages significantly inhibited glioblastoma growth without retarding the neural repair. We conclude that the surface functional group determines the properties as well as the interactions of C60 with NSCs and glioma cells, producing either a neuroprotective or antitumor effect for possible treatment of CNS-related diseases. KEYWORDS: water-soluble C60 fullerene derivatives, neural repair, glioblastoma, surface functionalization, antitumor

■

INTRODUCTION The global market of drugs for the central nervous system (CNS) is extremely underpenetrated. The current market for CNS drugs is about five times less than that for cardiovascular drugs.1 The main cause behind such underdevelopment is that many drugs do not have the ability to cross the human blood− brain barrier (BBB). Essentially, all large-molecule drugs are not likely to cross the BBB. Only a small portion (∼2%) of neuropharmacological agents with low molecular mass and high lipid solubility cross the BBB.2 Besides, only a few CNS diseases can respond to this small portion of small molecules constantly.3,4 Many neurological disorders (i.e., brain tumor, Alzheimer’s disease, and other CNS disorders) remain intractable to the conventional treatment of small-molecule pharmaceuticals. The difficulty and challenge are also implied by the fact that few pharmaceutical companies today possess a BBB drug-targeting project. With the advent of nanomedicine during recent years, tunable nanometer-sized devices have been suggested as a powerful tool that can offer a potential solution to the unmet medical need in enabling the drug to cross the BBB. Several © 2017 American Chemical Society

types of nanoparticles are utilized in biomedical research, such as gold nanoparticles, magnetic nanoparticles, water-soluble fullerene derivatives, and silica nanoparticles, among others. In particular, water-soluble C60 fullerene derivatives have attracted significant attention because of a broad range of biological activities discovered for these materials including potent antioxidant and radical scavengering properties as well as antimicrobial or antiviral activies.5 Some of the developed C60 fullerene compounds have the potential to be applied as imaging diagnostic agents and anticancer drugs.6−8 In addition, fullerene derivatives can penetrate efficiently through the BBB and can be used for drug delivery to brain. Therefore, design and investigation of the fullerene−drug hybrid systems might provide a promising approach to pharmacological therapy of neural disorders. Derivatives of C60 fullerene were shown to protect neurons from apoptosis which stimulated a commercial development of novel therapies for neurodegenerative disReceived: January 21, 2017 Accepted: March 6, 2017 Published: March 6, 2017 11482

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces

Figure 1. Molecular structures of the water-soluble fullerene C60 derivatives investigated.

■

eases.9−11 The aforementioned studies suggest that fullerene derivatives represent a promising group of compounds that can be useful in design of novel drugs for neurologic disorders. However, C60 fullerene is only sparingly soluble in some organic solvents and is completely insoluble in water. To solve this problem, chemical functionalization of carbon cage has to be performed. Changing the chemical structures of fullerene derivatives can be used to tailor their biological effects. This important aspect of medical chemistry of fullerenes remains unexplored. In the present study, we synthesized nine water-soluble C60 fullerene derivatives and evaluated their efficacy in repairing CNS damage and killing brain tumor. In vitro experiments were performed on neural stem cells (NSCs) and glioblastoma cells to select the most promising candidates. In vivo efficacy was assessed using the zebrafish model. We showed that surface functionalization of C60 might bring unique properties such as distinct regulations of the oxygen metabolism in target cells, producing either neuroprotective or apoptotic effects for therapy of CNS diseases and disorders.

RESULTS AND DISCUSSION The molecular structures of the water-soluble fullerene derivatives investigated are shown in Figure 1. General pathways used for their preparation are illustrated in Scheme S1. Compounds 1−3 bearing residues of phenylacetic, phenylpropanoic, and phenylbutyric acids, respectively, were synthesized according to the general approach previously reported by our group.12 Some spectral, physicochemical, and antiviral properties of 1−3 were presented more recently.13,14 Sulfur-containing fullerene derivatives 4 and 5 are novel compounds which were synthesized according to the previously reported procedure.15 It is noteworthy that compound 4 comprises five residues of the commercial drug captopril in its molecular framework. Captopril has been shown to exert neuroprotective effects in vitro and in vivo.16 Spectral properties of 4 and 5 are given in the Experimental Section. The synthesis and characterization of fullerene derivative 6 was previously reported by our group.17 Fullerene derivatives 7−9 bearing residues of such amino acids as phenyl alanine, serine, and βalanine, respectively, were synthesized following the previous report.18 The spectral properties of novel compound 9 (in the protected tert-butyl ester form) are also given in the Experimental Section. 11483

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces

Figure 2. Viability of neural stem cells (NSCs) and C6 glioblastoma cells treated with various functionalized fullerenes for 24 h. (A) The cell viability (and mitochondrial function) were evaluated for cells exposed to functionalized fullerenes 1−9 for 24 h. The cell viability for (B) NSCs and (C) C6 cells was determined by the MTT assay. Cell viability % = [(the mean optical density of the sample − blank)/(the mean optical density of the control − blank)] × 100%. *, p < 0.05, among the indicated groups. The concentrations of fullerene derivatives were 100 nM (for 1−6) and 50 mM (for 7−9).

μm in size.22 Similar behavior was experimentally observed for compounds 2 and 5. Considering these data, one can conclude that all investigated water-soluble fullerene derivatives represent nanoparticles composed of numerous individual molecules. These supramolecular architectures are bound by relatively weak van der Waals forces; therefore, they are supposed to be very labile and exist in a dynamic equilibrium with some amount of individual solvated molecules. Therefore, both nanoparticles and individual molecules are expected to exert different biological effects in the cells and animals. Some time ago there were active debates regarding the toxicity of fullerenes.23−25 More recent studies have shown that pristine C60 induces no significant toxic effects in vitro and in vivo.23,26 It is reasonable to assume that the toxicity of fullerene derivatives can depend significantly on the chemical structure of the appended organic addends and on the solubility of the compounds in aqueous media. We investigated the acute toxicity of compounds 1−9 in fibroblasts (L929) (Figure S2). The IC50 values of fullerene derivatives 1−3 to fibroblasts were estimated to be ∼400 nM. Compound 4 demonstrated an IC50 value exceeding 500 nM. More toxic were compounds 5 and 6, which displayed IC50 values of ∼200 nM. The least toxic were compounds 7−9 with IC50 ≈ 1−1.5 mM. We also examined the acute toxicity of compounds 1−9 in mice (intraperitoneal injection) and found that the fullerene derivatives bearing

It should be emphasized that compounds 1−9 bear five highly polar solubilizing groups attached at one semisphere of the fullerene cage, while the other semisphere remains nonfunctionalized and highly hydrophobic. On the one hand, such a unique molecular architecture distinguishes 1−9 from the vast majority of other known water-soluble fullerene derivatives. On the other hand, the aforementioned arrangement of organic addends on the carbon cage in compounds 1− 9 makes them truly amphiphilic and induces a strong tendency to self-assemble in polar solvents, especially water. Therefore, it is not surprising that aqueous solutions of 1−9 comprise relatively large nanoparticles rather than individual solvated molecules. Figure S1 and Table S1 give an overview of the aggregation behavior of fullerene derivatives 1−9. One might notice the presence of the smallest particles with the average hydrodynamic radius (⟨Rh⟩) close to 1 nm, which can be attributed to individual solvated molecules of 3. The observed medium-sized particles with ⟨Rh⟩ ≈ 6 nm might correspond to the small fullerene clusters such as micelles. The largest clusters with ⟨Rh⟩ ≈ 50 nm are represented, most probably, by double-layer fullerene vesicles. Similar vesicles were well-documented for a number of other amphiphilic fullerene derivatives.19−21 It is also notable that fullerene-based vesicles can also self-assemble to form larger supramolecular architectures approaching 1−100 11484

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces

Figure 3. Effect of various functionalized fullerenes on the mitochondrial metabolism and respiration of NSCs and C6 cells. (A) Expressions of specific mitochondrial related genes (HIF1α, Nrf-2, and AP1) examined by real-time RT-PCR at 24 h. The gene expression was normalized to GAPDH. (B) Mitochondrial respiration (basal OCR and ATP production) and nonmitochondrial oxygen consumption of NSCs and C6 cells treated with various functionalized fullerenes for 24 h. The oxygen consumption rate (OCR) is employed as a parameter to analyze the mitochondrial function. *, p < 0.05, among the indicated groups. The concentrations of fullerene derivatives were 100 nM (for 3−4) and 50 mM (for 7−8).

NSCs treated with 8 and 9 was significantly decreased. The viability of NSCs treated with 7 showed no significant change (Figure 2B). These results indicated that fullerene derivatives 1−6 promoted the survival and proliferation of brain neural progenitors. The viability of the C6 cells treated with phosphorus- and sulfur-containing compounds 4−6 was notably increased (by ∼130, ∼110, and ∼115%, respectively). On the contrary, compounds of 7−9 decreased the viability of C6 cells significantly. At the same time, 1−3 had no significant effect on the viability of C6 cells (Figure 2C). These results indicated that fullerene derivatives 7−9, bearing amino acid residues, inhibited the proliferation of C6 cells. It is interesting that compounds 1−3 bearing residues of phenylacetic, phenylpropanoic, and phenylbutiryc acids promoted the proliferation of NSCs but showed no effect on C6 cells. On the contrary,

aromatic carboxylic acid residues as solubilizing groups (compounds 1−3) demonstrated very low toxicity with LD50 values exceeding 300 mg/kg. Somewhat more toxic were compounds with sulfonic (5) and phosphonic (6) moieties, which are characterized by LD50 of 150−200 mg/kg. The least toxic compounds (4, 7, and 8) revealed outstanding LD50’s of 730, 680, and 725 mg/kg, respectively. Compound 9 has demonstrated somewhat higher toxicity with LD50 ∼ 350 mg/ kg. Summarizing these results, one might conclude that the investigated fullerene derivatives possess moderate to low toxicity and, therefore, are rather safe for different types of animal studies. We investigated the effects of water-soluble C60 fullerene derivatives on the survival and growth of NSCs and glioblastoma cells (C6 cells) (Figure 2). The viability of NSCs treated with functionalized fullerenes 1−6 for 24 h was increased considerably (∼130, ∼120, and ∼120%) and those of 11485

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces

Figure 4. Functional assay for the central nervous system (CNS) restoration in ethanol (EtOH)-induced CNS-impaired zebrafish larvae and braininjured adult zebrafish by treatment of various functionalized fullerenes. (A) Treatment procedures of developmental zebrafish embryos. (B) Spontaneous contraction determined at 18 somite stage of the larvae. (C) Coiling frequency (Hz) counted within 2 min at 24 hpf. (D) Hatching rate of the larvae determined at 48 hpf, which is an index for evaluation of the nervous function rescue. (E) Treatment procedures of adult zebrafish. (F) Percent functional recovery of the swimming speed for the adult zebrafish with various treatments. WT stands for wildtype. EtOH represents the defected group without any further treatment. *, p < 0.05, among the indicated groups. OB, Tel, OT, and CE represent olfactory bulb, telencephalon, optic tectum, and cerebellum. The concentrations of fullerene derivatives were 100 nM (for 3−4) and 50 mM (for 7−8).

neurodegenerative diseases and brain tumor formation,27 we examined whether water-soluble fullerene derivatives were able to induce mitochondria-associated cytoprotective antioxidant effects or antitumor effects. For this purpose, we examined the expression of the mitochondrial related markers HIF1α, Nrf-2, and AP-1 by real-time (quantitative) reverse transcriptionpolymerase chain reaction. The results are shown in Figure 3A. NSCs treated with compounds 3−7 expressed more mitochondrial related genes (HIF1α, Nrf-2, and AP-1) than did the control after 2 days. Among all C60 fullerene derivatives, the expression level of mitochondrial related genes was the highest for NSCs treated with compound 4. Meanwhile, the expression levels of mitochondrial-related genes of C6 cells were reduced after treatment with amino acid fullerene derivatives 7 and 8. Meanwhile, the expression of mitochondrial related genes was slightly increased for C6 cells treated with 3. These gene profiles revealed that functionalized fullerenes might be able to regulate the mitochondrial activity and exert either cytoprotective (antioxidant) or antitumor effects on different types of cells.

compound 7 with phenylalanine appendages inhibited C6 cells but had no effect on NSC survival. The effects of water-soluble fullerene derivatives on NSC differentiation are presented in Figure S3. Nestin is an intermediate filament protein that is expressed in undifferentiated CNS cells during development. It is a neural stem/ progenitor cell marker. The gene expression level of nestin after 7 days significantly increased (∼5 and ∼3.5 folds) for NSCs treated with 3 and 4 but significantly decreased for those treated with 1 and 2, as compared with the control. NSCs treated with fullerene derivatives 1−6 expressed more β-tubulin (a neural precursor marker) and MAP2 (a mature neuronal marker) than the control at 7 days. Among all C60 fullerene derivatives investigated, compound 4 induced the greatest expression levels of β-tubulin and MAP2. The above findings suggested that fullerene derivatives 1−6 might promote the neuronal differentiation of NSCs. Furthermore, compounds 3− 6 might sustain the self-renewal of some neuroprogenitors. Since mitochondria are critical regulators of cell death, proliferation, and differentiation and play a central role in 11486

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces

We also evaluated the potential of functionalized fullerenes in CNS repair for adult zebrafish. The recovery of movement (swimming) speed for the adult zebrafish is demonstrated in Figure 4E (n = 15 in each group). Brain-injured adult zebrafish were immobile or had imbalanced swimming. Animals injected with captopril-based fullerene derivative 4 showed 34, 57, and 72% recovery in locomotion after 2, 4, and 6 days, respectively. Meanwhile, animals injected with 3 showed 24, 41, and 63% recovery in locomotion after 2, 4, and 6 days, respectively. In contrast, the untreated group demonstrated no functional rescue even after 6 days. To assess the therapeutic feasibility of C60 fullerene derivatives in brain tumor in vivo, we evaluated the apoptotic activity of C6 cells in a tumor xenograft model. For that, zebrafish larvae (n = 90 in each group) were injected with C6 cells (2 × 102) and treated with the fullerene derivatives 3, 4, 7 and 8 after tumor grafting (Figure 5A). The obtained results (Figure 5B,C) showed that compounds 7 and 8 significantly reduced the tumor size. On the contrary, compound 4 even promoted the expansion of the tumor in comparison to the control. At the same time, compound 3, which had little effect on C6 growth in vitro, demonstrated no significant tumor size change in treated zebrafish. In addition, we demonstrated that the water-soluble C60 fullerene derivative could efficiently penetrate through BBB in mice (Figure S4). The results suggested that these water-soluble C60 fullerene derivatives may be used for drug delivery to brain. C60 fullerene derivatives, a unique category of materials with potent antioxidant properties, have been suggested to exert a broad range of biological activities including neuroprotective effects and anticancer properties.29 Many neurodegenerative disorders such as Alzeimer’s and Parkinson’s are connected to the excessive production of nitric oxide and oxygen radical species. In addition, reactive oxygen species (ROS) are associated with the pathology of many acute brain injuries.30 The neuroprotective capacity of fullerenes is attributed to their reactivity with ROS including hydroxyl and superoxide free radicals.31 Literature data have suggested that C60(OH)n may be an excellent antioxidant absorbing ROS and reducing apoptosis in vitro.31,32 Carboxyfullerenes have also demonstrated excellent antioxidative efficacy for preventing neurodegeneration involved in the amyotrophic lateral sclerosis.33 According to these studies, C60 fullerene derivatives possess neuroprotective effects at the cellular level. However, no study has examined the neurophysiological brain functions after treatment with fullerene derivatives in vivo. In the present work we investigated representative examples of several different chemical classes of water-soluble fullerene derivatives. First, we studied compounds with different types of linkages between the fullerene cage and the solubilizing addend: C−C bonds (compounds 1−3), C−S bonds (compounds 4−5), C−P bonds (compound 6), and C−N bonds (compounds 7−9). Second, we utilized solubilizing groups of different natures: The vast majority of compounds (1−4 and 7−9) bear carboxylic groups (−COO −K+); compounds 4 and 5 comprise sulfonic (−SO3−K+) and phosphonic (−PO(O−K+)2) residues in the form of potassium salts. Third, the chemical structure of spacers between the carbon sphere and terminal solubilizing groups was altered. For instance, in the group of C−C compounds 1−3, we increased systematically the number of CH2 units in the aliphatic chain. In the group of C−N compounds, we utilized different amino acids as addends: phenyl alanine (7), serine (8), and beta-

In order to directly evaluate the mitochondrial function in NSCs and C6 cells under the influence of functionalized fullerenes, a bioenergetic assay was used to measure the cellular energetic oxygen consumption rate (OCR). OCR is a parameter to examine the mitochondria and is an index for normal cell function. In cancer cells, it serves as an indicator for the metabolic switch from the healthy oxidative phosphorylation to aerobic glycolysis. Results of the bioenergetic measurement are presented in Figure 3B. The basal OCR and adenosine triphosphate (ATP) production were significantly induced in NSCs treated with 3 and 4. Nonmitochondrial oxygen consumption was not significantly altered by the treatment of cells with 3 and 4. Treating C6 cells with amino acid fullerene derivatives 7 and 8 for 24 h resulted in decreases in the basal OCR, ATP production, and nonmitochondrial oxygen consumption. Taken together, these results indicated that fullerene derivatives 3 and 4 specifically increased mitochondrial function to induce NSC proliferation and differentiation. At the same time, compounds 7 and 8 were able to destroy the mitochondrial function and nonmitochondrial respiration, leading to the antitumor response. Zebrafish (Danio rerio) is an important model for studying neuroscience. Zebrafish possess a BBB that is functionally homologous to that of humans.28 The exposure of developing zebrafish larvae to ethanol (EtOH, 1.5%) can result in CNS deficits (Figure 4A). The spontaneous coiling contraction of the zebrafish larvae (earliest motor behavior) was analyzed at 18 stage (Figure 4B, n = 90 per group). Wild-type larvae not immersed in EtOH (blank control) demonstrated ∼85% full response (contraction from one side to another side) and ∼15% slight response (single-side contraction or very small coiling angle). The CNS-impaired larvae without any further treatment demonstrated ∼76% no contraction and ∼24% slight contraction. CNS-impaired larvae incubated with compound 7 showed ∼28.6% full response, ∼43% slight contraction, and ∼28.4% no contraction. Larvae incubated with compound 8 showed ∼10% slight contraction and ∼90% no contraction. Most remarkably, larvae incubated with compound 3 and sulfur-containing fullerene derivatives 4−5 showed ∼70, 55, and 65% full response, respectively. The frequency of coiling contraction detected at 24 h postfertilization (hpf) is shown in Figure 4C. The coiling frequency of wild-type larvae was ∼0.075 Hz. The coiling frequency of the group treated with 7 was slightly increased to ∼0.05 Hz. Surprisingly, treatment of embryos with compound 8 decreased coiling frequency even further down to 0.01 Hz. The coiling frequencies for the groups of embryos treated with 3−5 were significantly increased to ∼0.065, ∼0.10, and ∼0.15 Hz, respectively. The lowest coiling frequency (∼0.03 Hz) was observed in the untreated impaired group. Hatching rate is the most essential index to evaluate the functional rescue in the experimental model of EtOH-induced CNS deficit. As shown in Figure 4D, wild-type larvae had a 95% hatching rate. The untreated impaired group showed a hatching rate of 17%. The group incubated with compound 7 revealed a hatching rate of 25%. Meanwhile, the group incubated with compound 8 demonstrated no larvae hatching from eggs. The group incubated with the captopril-based fullerene derivative 4 showed the best hatching rate of ∼65%, while those treated with 3 and 5 showed hatching rates of ∼55%. The CNS-injury model showed that fullerene derivatives 3−5 could restore the function of injured nervous system in the larval period. 11487

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces

addition, they may affect the mitochondrial membrane potential, availability of the substrates (e.g., NADH and FADH2/succinate) for respiration, and ATP synthesis.37 The upregulation of mitochondrial gene expression and respiration found for the NSCs treated with functionalized fullerenes 1−6 indicated that these functionalized fullerenes had mediated protection against mitochondrial-derived ROS and enhanced NSC survival and proliferation to resume the CNS function. C60 fullerene derivatives have been developed for cancer theranostics by magnetic resonance imaging and photoacoustic imaging.38 Gadolinium endohedral metallofullerenol (Gd@ C82(OH)n) and C60(OH)n prevented the growth of murine hepatoma effectively.38 No study has examined the potential of C60 fullerene derivatives as cancer chemotherapeutics with respect to the brain cancer therapy. The current study was the first to show that C60 fullerene derivatives (7−9) efficiently inhibited the growth and invasion ability of glioblastoma. Prior literature data revealed that HIF-1α, Nrf2, and AP-1 were overexpressed in glioblastoma,36 and the overexpression of these genes may promote tumor growth, metastasis, and resistance to radioactive therapies through their role in regulating mitochondrial respiration to overcome hypoxia.36 Our study demonstrated that the antitumor mechanism of functionalized fullerenes 7−9 might be related to their ability to regulate the oxidative stress in tumor-bearing animals. Meanwhile, we observed that functionalized fullerene 7 had no effect on NSCs and thus may be particularly useful for anticancer therapy. The mediation of oxidation stress in glioblastoma cells by the functionalized C60 derivatives might increase the feasibility of using these compounds to cancer chemotherapeutics. Taken together, we showed that the surface functional group determines the properties as well as the interactions of C60 with NSCs and glioma cells. We suggested that the different terminal solubilizing groups may affect the stereochemical structure, solubility, size, and self-assembly ability of water-soluble fullerene derivatives. These properties may affect the function of those compounds (compounds 1− 9). However, all interactions of fullerene derivatives with the biological targets are mediated by electrostatic and hydrogen bonding. Therefore, the number of organic addends on the fullerene sphere, their polarity, and the presence of ionic groups (like COO−) should affect the binding of the fullerene derivatives to the biological targets. In summary, compound 3 can promote NSC proliferation through regulating the mitochondrial activity but does not exert a proliferative effect on glioblastoma. Moreover, compound 7 can kill glioblastoma cells through regulating the mitochondrial activity but does not produce a destructive effect on NSCs. Comparing these results, we assume that fine-tuning the chemistry of C60 fullerene derivatives should allow one to enhance a major therapeutic function and reduce the side effects. As illustrated in Figure 6, the fullerene derivatives with different functional groups act on mitochondria like molecular switches that can either provoke or suppress the oxidation to achieve neuroprotective or antitumor effects in different types of cells.

Figure 5. Morphology of glioblastoma in the brain of zebrafish larvae treated with various functionalized fullerenes. (A) PKH26 (red fluorescence)-labeled C6 cells were injected into the brain of each Tg (Fli-EGFP) zebrafish larva. The zebrafish fli promoter can drive the expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. After 1 day, zebrafish larvae were incubated with fullerene derivatives for 3 days post-treatment (dpt) and determined the tumor volume by the ImageJ software. (B) Morphology for glioblastoma in Tg (Fli-EGFP) zebrafish larvae. Each brain tumor-bearing larval group was treated with fullerene derivatives 3, 4, 7, and 8 at 48 h postfertilization (hpf). The fluorescence microscopy images of Tg (Fli-EGFP) zebrafish larvae were taken at 0 and 3 dpt. (C) Tumor growth rate for brain tumor-bearing larvae treated with various functionalized fullerenes. The tumor growth rate was determined as percent of the tumor volume at 3 dpt relative to the tumor volume at 0 dpt. The labels fb, mb, and hb represent forebrain, midbrain, and hindbrain. The concentrations of fullerene derivatives were 100 nM (for 3−4) and 50 mM (for 7−8).

alanine (9). The results of the performed experiments showed that both linkage type and, particularly, the nature of the solubilizing groups affect the toxicity of the water-soluble fullerene derivatives. Indeed, compounds 5 and 6, bearing sulfonic and phosphonic residues, respectively, showed considerably lower semilethal doses LD50 compared to those of the rest of fullerene derivatives bearing carboxylic groups. All the other biological effects of the fullerene derivatives revealed in this study depended mostly on the type of linkages between the fullerene cage and the solubilizing groups. The study reported herein demonstrated that fullerene derivatives 1−6 could increase the proliferation/survival of NSCs and function as protective agents against the acute traumatic injury induced degeneration of brain in vivo. Transcription factors HIF1α, Nrf-2, and AP-1 are increasingly being recognized as crucial regulators of ROS production by mitochondria.34−36 In

■

CONCLUSION Water-soluble C60 fullerene derivatives with different types of linkages between the fullerene cage and the solubilizing addend could exert different effects on neural stem cells and glioblastoma cells. The compound bearing residues of phenylbutiryc acids significantly induced neural repair without causing 11488

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces

obtained solid was dissolved in a 0.025 M solution of K2CO3 (130 mL), filtered through a tight paper filter (blue tape), and then through a PES syringe filter (average pore size 0.45 μm). The filtrate was quenched by addition of 1 M hydrochloric acid (15 mL), which produced orange flaky precipitate. The precipitate was collected by centrifugation, washed 3 times with deionized water, and dried in vacuum at room temperature. Target acid 4 was obtained as a red solid in 68% yield. The full potassium salt of 4 was obtained by dissolving the acid in a stoichiometric amount of 0.025 M K2CO3 followed by freeze-drying of the solution. 1H NMR((CD3)2CO:CS2 (1:1), 600 MHz, δ, ppm) 1.15−1.26 (m, 15H, CH3), 1.76−2.34 (m, 20H, CH2), 2.53−2.97 (m, 10H, CH2−S, CH−C(O)), 3.1−3.23 (m, 5H, CH2−S), 3.32−3.78 (m, 10H, CH2−N), 4.15−4.33 (m, 5H, CH−COOH), 4.95 (s, 1H, C60−H). 13C NMR ((CD3)2CO:CS2 (1:1), 150 MHz, δ, ppm) 16.54 (CH3), 16.65 (CH3), 24.37 (CH2), 24.42 (CH2), 27.08 (CH2), 28.72 (CH2), 41.14 (CH−C(O)), 41.33 (CH−C(O)), 46.44 (S-CH2), 46.50 (S−CH2), 46.55 (S−CH2), 54.00 (cage sp3C), 55.92 (cage sp3C), 56.60 (cage sp3C), 58.26 (CH−COOH), 58.30 (CH−COOH), 58.34 (CH−COOH), 59.06 (cage sp3C−H), 140.06, 141.95, 142.23, 142.48, 142.58, 142.61, 142.72, 143.27, 143.65, 143.76, 144.07, 145.68, 146.12, 146.16, 146.22, 146.27, 146.31, 146.35, 147.45, 147.51, 147.53, 147.65, 147.73, 147.81, 147.87, 147.99, 148.17, 148.20, 172.72 (N− CO), 173.25 (N−CO), 173.30 (COOH), 173.34(COOH), 173.45 (COOH). ESI MS: m/z = 1800 ([M − H]−), 900 ([M − H]2−). Synthesis of Compound 5. Chlorofullerene C60Cl6 (1000 mg, 1.07 mmol) was dissolved in toluene (800 mL) under intense stirring. A solution of sodium 3-mercaptopropane-1-sulfonate (1.9 g, 10.7 mmol) and equimolecular amount of 15-crown-5 in 100 mL of acetonitrile was added in one portion, and the mixture was stirred further for 5 min at room temperature. The formed organge-red precipitate was collected by filtration, washed with acetone (3 × 100 mL) and dried in air. The obtained solid was dissolved in deionized water (150 mL), filtered through a tight paper filter (blue tape) and then through a PES syringe filter (average pore size 0.45 μm). The filtrate was subjected to freeze-drying to obtain the target salt 5 as a light orange powder. Isolated yield of 5 was 84%. 1H NMR (D2O, 500 MHz, δ, ppm) 2.00−2.19 (m, 10H), 2.87−2.91 (m, 6H), 2.93−3.00 (m, 4H), 3.08−3.12 (m, 6H), 3.20−3.23 (m, 4H), 3.45 (c, 1H). 13C NMR (D2O, 125 MHz, δ, ppm) 23.02 (CH2), 24.50 (CH2), 30.39 (CH2), 30.64 (CH2), 31.13 (CH2), 48.27 (CH2), 48.84 (CH2), 52.50 (cage sp3C), 54.44 (cage sp3C), 55.03 (cage sp3C), 59.38 (cage sp3C), 141.67, 141.72, 141.78, 141.88, 142.41, 142.53, 142.58, 142.79, 143.16, 143.58, 143.71, 144.99, 145.20, 145.27, 145.42, 145.99, 146.36, 146.52, 146.71, 146.92, 147.02, 147.16, 148.96, 149.70, 152.00, 152.15, 152.66, 152.88. FTIR (KBr pellet, ν, cm−1) 454.00 (W), 498.00 (W), 528.00 (M), 542.00 (M), 558.00 (W), 606.00 (M), 668.00 (W), 738.00 (W), 808.00 (VW), 862.00 (VW), 944.00 (W), 1052.00 (VS), 1114.00 (M), 1190.00 (VS), 1350.00 (W), 1418.00 (W), 1634.00 (M), 1700.00 (W). Synthesis of Compound 9. Chlorofullerene C60Cl6 (187 mg, 0.2 mmol) was dissolved in toluene (200 mL) under inert atmosphere (argon). Large excess of anhydrous potassium carbonate (1.0 g) was added in one portion followed by a dropwise addition of a solution of tert-butyl ester of β-alanine (174.2 mg, 1.2 mmol) in 20 mL of toluene. After completion of the reagent addition, the mixture was stirred in argon at room temperature. The course of the reaction was monitored by HPLC. The appearance of a single dominating component and disappearance of C60Cl6 was observed approximately in 2−3 h. The reaction mixture was separated from inorganic solids by filtration and then poured at the top of silica gel column. Elution with toluene followed by toluene-acetonitrile (96:4 v/v) mixture produced the bright orange-red fraction of the target compound, which was concentrated to dryness at the rotary evaporator, washed with acetonitrile and hexanes and dried in air. Target tert-butyl ester of 9 was obtained as a dark red solid with 57% yield. Removal of tertbutoxycarbonyl groups and dissolving the resulting acid in a stoichiometric amount of 0.025 M K2CO3 produced water-soluble potassium salt 9 which was used in biological assays. 1H NMR (CDCl3, 600 MHz, δ, ppm) 1.50 (m, 45H, C(CH3)3), 2.67−2.75 (m, 10H, CH2−N), 3.50−3.62 (m, 10H, CH2−C(O)). 13C NMR (CDCl3,

Figure 6. Fullerenes with different functional groups act on mitochondria that can either induce or reduce the oxidation to achieve neuroprotective or antitumor effects in different types of cells. (A) Compounds 3 and 4 significantly promoted the NSC proliferation and neural repair through increasing mitochondrial function. (B) Compounds 7 and 8 could destroy the mitochondrial function, leading to the antitumor response.

tumor growth. Meanwhile, the compound with phenylalanine appendages significantly inhibited the glioblastoma growth without retarding the neural repair. These seemly opposite effects were correlated with the mitochondria metabolic changes upon exposure to these fullerene derivatives. Moreover, in vivo experiments suggested that the water-soluble fullerene may cross the BBB and function on the brain as neuroprotective or antitumor agents, which are potential brain medicine in the future.

■

EXPERIMENTAL SECTION

Synthesis of Compounds 1−3 and 6−8. Synthesis and spectral characteristics of water-soluble fullerene derivatives 1−3 and 6−8 were reported previously.12−15 Synthesis of Compound 4. Chlorofullerene C60Cl6 (1000 mg, 1.07 mmol) was dissolved in toluene (800 mL) under intense stirring. A solution of captopril (2.5 g, 11.5 mmol) and N,N-diisopropylethylamine (826 mg, 6.4 mmol) in toluene (20 mL)/acetonitrile (20 mL) mixture was added in one portion, and the mixture was stirred further for 5 min at room temperature. The formed red-brown precipitate was collected by filtration, washed with hexanes, and dried in air. The 11489

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces 150 MHz, δ, ppm) 28.20 (C(CH3)3), 28.23 (C(CH3)3), 28.25 (C(CH3)3), 36.07 (CH2−CH2−N), 36.18 (CH2−CH2−N), 36.93 (CH2−CH2−N), 42.54 (CH2−N), 42.86 (CH2−N), 44.61 (CH2−N), 65.32 (cage sp3C), 67.73 (cage sp3C), 65.59 (cage sp3C), 80.54 (O− C), 80.68 (O−C), 80.70 (O−C), 140.30, 142.57, 143.19, 143.58, 143.89, 144.06, 144.19, 144.23, 144.41, 144.49, 144.85, 145.15, 145.48, 147.21, 147.24, 147.31, 147.90, 148.35, 148.40, 148.45, 148.71, 148.88, 149.71, 150.87, 154.04, 154.14, 171.93 (COOBut), 172.19 (COOBut), 172.21 (COOBut). ESI MS: m/z = 1442 ([M − Cl]−). Culture of Murine Neural Stem Cells and Rat Glioblastoma Cells (C6 Cells). Murine neural stem cells (NSCs) were obtained from the hippocampus of adult mouse brain and introduced with the promoter F1B-green fluorescence protein (F1B-GFP).39,40 These cells were maintained in Dulbecco’s modified Eagle’s medium and Ham’s F12 (DMEM/F12, Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 400 μg/mL geneticin (Invitrogen), and 100 U/mL pen−strep mixtures (Caisson Laboratories, USA). Cultures were incubated in a humidified incubator with 5% CO2 at 37 °C. The culture medium was refreshed every 2 days. C6 cells were cultured in DMEM containing 10% fetal bovine serum, 50 U/mL pen−strep mixtures, and 1% sodium pyruvate, and incubated in a humidified incubator with 5% CO2 at 37 °C. The culture medium was refreshed every 2 days. Cell Viability Assay. Cell viability was analyzed using the MTT assay. NSCs or C6 cells (1 × 104 in number) were seeded in 96-well plates and treated with fullerene derivatives 1−6 (100 nM) and fullerene derivatives 7−9 (50 mM) in cell culture medium. Following incubation for 24 h, the cell viability was measured. Tetrazolium dye (100 μL, 0.5 mg/mL, Sigma) was added to each well and the cells incubated at 37 °C for 4 h in a humidified chamber, then removed at the end of the incubation period. The formazan formed was completely dissolved by DMSO, and the absorbance was read at 570 nm by a UV/vis plate reader (SpectraMax M5, USA). Cell viability was determined as percent of viable cells relative to an untreated control. Results were the mean values and standard deviation (SD) from at least three different experiments in triplicate. Gene Expression Analysis. Total RNA was extracted from NSCs and C6 cells that were treated with fullerene C60 derivates using standard protocol (Invitrogen, USA) and resuspended in nuclease-free water. The concentration and purity of RNA were measured with a spectrophotometer (NanoDrop Technologies). RNA strand was first reverse transcribed into cDNA and amplified using the Thermoscript RT-PCR system (Invitrogen) priming with random hexamers. The real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using the DyNAmo Flash SYBR Green qPCR Kit (Finnzymes Oy, Espoo, Finland). The PCR reaction and detection were placed in a StepOnePlus Real-Time PCR System (Applied Biosystems, USA). The expression was represented as the relative ratio to GAPDH. The primer sequences for each gene used in this study are demontrated in Table S2. Cell Labeling. The PKH26 (Red Fluorescence Cell Linker Kit, Sigma) was used to label C6 cells for cell tracking in animal studies. Cells (at a density of 1 × 107 cells/mL) were labeled by mixing with 2 × 10−6 M PKH26, which could stably incorporate into the cell membrane with its long aliphatic tails. The labeling process was stopped with complete medium. The labeled cells were washed and ready for use. Zebrafish Maintenance. Wild-type (AB line) and Fli-EGFP transgenic zebrafish (of which the fli promoter can drive the expression of enhanced green fluorescent protein in all blood vessels throughout embryogenesis) larvae were purchased from the Zebrafish International Resource Center (Oregon, USA) and were raised, maintained, and paired under standard conditions. All procedures involving zebrafish work and tests in this study followed the ethical guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC). Zebrafish larvae were maintained in a Petri dish with larval water (5 mM NaCl, 0.17 mM KCl, 0.33 mM MgSO4, and 0.33 mM CaCl2) at 34 °C.41 Adult zebrafish were kept under a 14 h/ 10 h light−dark cycle at room temperature. The body length of all adult zebrafish was in the range of 2.8−3.8 cm in this study.

Functional Assay for the Zebrafish CNS Repair. In vivo CNS repair was evaluated by treatment with fullerene C60 derivates in the neural deficit model of zebrafish larvae. These larvae were divided into seven groups in this study. The first group was larvae in normal larval water as a blank control. The other groups were immersed in larval water with 1.5% ethanol (EtOH) for 4 h to impair the nervous system. The medium was later replaced with fresh larval water containing various compounds including treatment with fullerene derivatives 3−5 (100 nM) and fullerene derivatives 7 and 8 (0.5 mM) in the larval water for 12 h before incubation in fresh normal larval water. The untreated control was that group without any treatment. All experiments were performed on multiple samples (n = 30) and repeated by three independent experiments. Larval movement behaviors were observed by a dissecting microscope. Video images were captured by an charge-coupled device (Olympus C-7070 CCD). Rescue of the neural deficit by various treatments was evaluated by the coiling contraction and hatching rate (an index of motor function and CNS function). For in-chorion coiling contraction, the total contraction in 3 min at 24 hpf was counted and demonstrated as the number of times per second (Hz). The hatching rate of the larvae was evaluated at 58 hpf. The values were compared to those of the blank control to reveal the neural functional rescue by different treatments. Adult zebrafish were separated into four groups in this study. The blank control was the zebrafish without an injury in the cerebellum. In the other groups, an injury was created in the zebrafish in the cerebellum by a syringe needle (27G; Thermo Scientific, USA).42 The treatments of animals were injected compounds 3 and 4 (100 nM) into cerebellum of zebrafish. All tests were performed on multiple samples (n = 15) in at least three different experiments in triplicate. Zebrafish locomotion analysis was examined for animals up to 6 days before the zebrafish were sacrificed. These zebrafish were trained to move in a tank (260 mm × 90 mm × 50 mm). The moving speed was analyzed with ImageJ software. The recovery (%) of motor function was calculated from the following equation: recovery (%) = (moving speed of the treated or untreated experiment group/moving speed of the blank control group) ×100%. Cancer Cell Transplantation in Zebrafish Larvae. For zebrafish xenotransplantation, 24 hpf Fli-EGFP transgenic zebrafish strains of transgenic zebrafish larvae were dechorionated and anaesthetized in E3 medium supplemented with phenythiourea (PTU, Sigma-Aldrich) and 0.04 mg/mL tricaine (Sigma-Aldrich) before the injection of C6 cells. PKH26-labeled C6 cells were injected into the brain of each larva. After that, the zebrafish were kept in E3 medium supplemented with PTU at 28 °C for 1 h. After a visible cell mass at the injection site was obseved, the zebrafish were transferred to the incubator which was maintained at 34 °C. After 1 day, zebrafish larvae were incubated with fullerene derivatives 3 and 4 (100 nM) and fullerene derivatives 7 and 8 (0.5 mM) in E3 medium containing PTU for 3 dpt at 34 °C. The tumor volume was determined by ImageJ software (National Institutes of Health, USA). The growth rate of tumor was determined at 3 dpt as percent of the tumor volume at 3 dpt relative to that at 0 dpt. Metabolic Flux Analysis by Seahorse XF-96. The oxygen consumption rate (OCR) was measured at 37 °C using an XF96 extracellular analyzer (Seahorse Bioscience). NSCs (2 × 104 cells/ well) were plated in 8-well plates and incubated with fullerene derivatives 3 and 4 (100 nM) in cell culture medium. C6 cells (4 × 104 cells/well) were seeded in 8-well plates and incubated with fullerene derivatives 7 and 8 (50 mM) in cell culture medium. Following 24 h incubation, the medium was switched to unbuffered DMEM (DMEM added with 25 mM glucose, 1 mM sodium pyruvate, 31 mM NaCl, 2 mM GlutaMax, pH 7.4) and incubated at 37 °C without 5% CO2 for 1 h. On the day of assay, injection reagents were adjusted to pH 7.4. The OCR was measured and computed by the Seahorse XF-96 software automatically. Every value represents an average of 3−6 different wells. Statistical analysis. Data from the experiments were presented as mean ± standard deviation. Reproducibility of each experiment in vitro and in vivo was confirmed independently three times. Statistical differences among the experimental groups were determined by one11490

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces way ANOVA. Results were considered statistically significant when p value < 0.05.

■

(7) Xiao, L.; Takada, H.; Maeda, K.; Haramoto, M.; Miwa, N. Antioxidant Effects of Water-Soluble Fullerene Derivatives Against Ultraviolet Ray or Peroxylipid through Their Action of Scavenging the Reactive Oxygen Species in Human Skin Keratinocytes. Biomed. Pharmacother. 2005, 59, 351−358. (8) Xiao, L.; Takada, H.; Gan, X.; Miwa, N. The Water-Soluble Fullerene Derivative ″Radical Sponge″ Exerts Cytoprotective Action against UVA Irradiation but Not Visible-light-catalyzed Cytotoxicity in Human Skin Keratinocytes. Bioorg. Med. Chem. Lett. 2006, 16, 1590− 1595. (9) Dugan, L. L.; Turetsky, D. M.; Du, C.; Lobner, D.; Wheeler, C.; Almli, C. R.; Shen, C.K.-F.; Luh, T. Y.; Choi, D. W.; Lin, T. S. Carboxyfullerenes as Neuroprotective Agents. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 9434−9439. (10) Ali, S. S.; Hardt, J. I.; Dugan, L. L. SOD Activity of Carboxyfullerenes Predicts Their Neuroprotective Efficacy: a Structure-activity Study. Nanomedicine 2008, 4, 283−294. (11) Ali, S. S.; Hardt, J. I.; Quick, K. L.; Kim-Han, J. S.; Erlanger, B. F.; Huang, T. T.; Epstein, C. J.; Dugan, L. L. A Biologically Effective Fullerene (C60) Derivative with Superoxide Dismutase Mimetic Properties. Free Radical Biol. Med. 2004, 37, 1191−1202. (12) Troshina, O. A.; Troshin, P. A.; Peregudov, A. S.; Kozlovskiy, V. I.; Balzarini, J.; Lyubovskaya, R. N. Chlorofullerene C60Cl6: a Precursor for Straight Forward Preparation of Highly Water-soluble Polycarboxylic Fullerene Derivatives Active against HIV. Org. Biomol. Chem. 2007, 5, 2783−2791. (13) Fedorova, N. E.; Klimova, R. R.; Tulenev, Y. A.; Chichev, E. V.; Kornev, A. B.; Troshin, P. A.; Kushch, A. A. Carboxylic Fullerene C60 Derivatives: Efficient Microbicides against Herpes Simplex Virus and Cytomegalovirus Infections in Vitro. Mendeleev Commun. 2012, 22, 254−256. (14) Kobzar, O. L.; Trush, V. V.; Tanchuk, V. Y.; Voronov, I. I.; Peregudov, A. S.; Troshin, P. A.; Vovk, A. I. Polycarboxylic Fullerene Derivatives as Inhibitors of Protein Tyrosine Phosphatases. Mendeleev Commun. 2015, 25, 199−201. (15) Khakina, E. A.; Yurkova, A. A.; Peregudov, A. S.; Troyanov, S. I.; Trush, V. V.; Vovk, A. I.; Mumyatov, A. V.; Martynenko, V. M.; Balzarini, J.; Troshin, P. A. Highly Selective Reactions of C60Cl6 with Thiols for the Synthesis of Functionalized [60]Fullerene Derivatives. Chem. Commun. 2012, 48, 7158−7160. (16) Sonsalla, P. K.; Coleman, C.; Wong, L. Y.; Harris, S. L.; Richardson, J. R.; Gadad, B. S.; Li, W.; German, D. C. The Angiotensin Converting Enzyme Inhibitor Captopril Protects Nigrostriatal Dopamine Neurons in Animal Models of Parkinsonism. Exp. Neurol. 2013, 250, 376−383. (17) Yurkova, A. A.; Khakina, E. A.; Troyanov, S. I.; Chernyak, A.; Shmygleva, L.; Peregudov, A. S.; Martynenko, V. M.; Dobrovolskiy, Y. A.; Troshin, P. A. Arbuzov Chemistry with Chlorofullerene C60Cl6: a Powerful Method for Selective Synthesis of Highly Functionalized [60]Fullerene Derivatives. Chem. Commun. 2012, 48, 8916−8918. (18) Kornev, A. B.; Khakina, E. A.; Troyanov, S. I.; Kushch, A. A.; Peregudov, A.; Vasilchenko, A.; Deryabin, D. G.; Martynenko, V. M.; Troshin, P. A. Facile Preparation of Amine and Amino Acid Adducts of [60]Fullerene Using Chlorofullerene C60Cl6 as a Precursor. Chem. Commun. 2012, 48, 5461−5463. (19) Zhou, S.; Burger, C.; Chu, B.; Sawamura, M.; Nagahama, N.; Toganoh, M.; Hackler, U. E.; Isobe, H.; Nakamura, E. Spherical Bilayer Vesicles of Fullerene-Based Surfactants in Water: a Laser Light Scattering Study. Science 2001, 291, 1944−1947. (20) Zhou, S.; Ouyang, J.; Golas, P.; Wang, F.; Pan, Y. Structural Study of the Self-assembled Fullerene Carboxylates: Monoadducts versus Bisadducts. J. Phys. Chem. B 2005, 109, 19741−19747. (21) Partha, R.; Lackey, M.; Hirsch, A.; Casscells, S. W.; Conyers, J. L. Self Assembly of Amphiphilic C60 Fullerene Derivatives into Nanoscale Supramolecular Structures. J. Nanobiotechnol. 2007, 5, 6. (22) Burger, C.; Hao, J.; Ying, Q.; Isobe, H.; Sawamura, M.; Nakamura, E.; Chu, B. Multilayer Vesicles and Vesicle Clusters Formed by the Fullerene-Based Surfactant C60(CH3)5K. J. Colloid Interface Sci. 2004, 275, 632−641.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01077. Scheme for the preparation of water-soluble fullerene derivatives, aggregation behavior of compounds 1−9, acute toxicity of compounds 1−9 in fibroblasts (L929), effects of water-soluble fullerene derivatives on NSC differentiation, and animal brain images (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +886-2-3366-5313. Fax: +886-2-3366-5237. Institute of Polymer Science and Engineering, National Taiwan University, No. 1, Sec. 4 Roosevelt Road, Taipei 10617, Taiwan, R.O.C. *E-mail: [email protected]. Phone: +7-496-522-14-18. ORCID

Shan-hui Hsu: 0000-0002-3420-7662 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the bilateral Taiwanese-Russian research project (RFBR No. 16-53-52030; MOST 105-2923-E002−003 -MY3). We are obliged to Dr. Ing-Ming Chiu (Institute of Cellular and Systems Medicine, National Health Research Institutes, Zhunan, Taiwan) for providing murine NSCs. We also appreciate the Taiwan Zebrafish Core Facility located in National Taiwan University (NTU-ERP-104R8600) for the technical and facility supports.

■

ABBREVIATIONS CNS, central nervous system BBB, blood−brain barrier ATP, adenosine triphosphate ORC, oxygen consumption rate EtOH, ethanol MRI, magnetic resonance imaging

■

REFERENCES

(1) Pardridge, W. M. Why Is the Global CNS Pharmaceutical Market so Under-Penetrated? Drug Discovery Today 2002, 7, 5−7. (2) Pardridge, W. M. Brain Drug Targeting and Gene Technologies. Jpn. J. Pharmacol. 2001, 87, 97−103. (3) Lipinski, C. A. Drug-like Properties and the Causes of Poor Solubility and Poor Permeability. J. Pharmacol. Toxicol. Methods 2000, 44, 235−249. (4) Ghose, A. K.; Viswanadhan, V. N.; Wendoloski, J. J. A Knowledge-Based Approach in Designing Combinatorial or Medicinal Chemistry Libraries for Drug Discovery. 1. A Qualitative and Quantitative Characterization of Known Drug Databases. J. Comb. Chem. 1999, 1, 55−68. (5) Schnorr, J. M.; Swager, T. M. Emerging Applications of Carbon Nanotubes. Chem. Mater. 2011, 23, 646−657. (6) Xiao, L.; Aoshima, H.; Saitoh, Y.; Miwa, N. The Effect of Squalane-Dissolved Fullerene-C60 on Adipogenesis-Accompanied Oxidative Stress and Macrophage Activation in a PreadipocyteMonocyte Co-Culture System. Biomaterials 2010, 31, 5976−5985. 11491

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492

Research Article

ACS Applied Materials & Interfaces (23) Sayes, C. M.; Fortner, J. D.; Guo, W.; Lyon, D.; Boyd, A. M.; Ausman, K. D.; Tao, Y. J.; Sitharaman, B.; Wilson, L. J.; Hughes, J. B.; West, J. L.; Colvin, V. L. The Differential Cytotoxicity of WaterSoluble Fullerenes. Nano Lett. 2004, 4, 1881−1887. (24) Bal, P. Research Highlights. Nature 2004, 431, 756−756. (25) Gharbi, N.; Pressac, M.; Hadchouel, M.; Szwarc, H.; Wilson, S. R.; Moussa, F. [60] Fullerene Is a Powerful Antioxidant in Vivo with No Acute or Subacute Toxicity. Nano Lett. 2005, 5, 2578−2585. (26) Usenko, C. Y.; Harper, S. L.; Tanguay, R. L. In Vivo Evaluation of Carbon Fullerene Toxicity Using Embryonic Zebrafish. Carbon 2007, 45, 1891−1898. (27) Carew, J. S.; Huang, P. Mitochondrial Defects in Cancer. Mol. Cancer 2002, 1, 9. (28) Xie, J.; Farage, E.; Sugimoto, M.; Anand-Apte, B. A Novel Transgenic Zebrafish Model for Blood-Brain and Blood-Retinal Barrier Development. BMC Dev. Biol. 2010, 10, 76. (29) Bakry, R.; Vallant, R. M.; Najam-ul-Haq, M.; Rainer, M.; Szabo, Z.; Huck, C. W.; Bonn, G. K. Medicinal Applications of Fullerenes. Int. J. Nanomedicine. 2007, 2, 639−649. (30) Kim, G. W.; Kondo, T.; Noshita, N.; Chan, P. H. Manganese Superoxide Dismutase Deficiency Exacerbates Cerebral Infarction after Focal Cerebral Ischemia/Reperfusion in Mice: Implications for the Production and Role of Superoxide Radicals. Stroke 2002, 33, 809− 815. (31) Liu, Q.; Zhang, X.; Zhang, X.; Zhang, G.; Zheng, J.; Guan, M.; Fang, X.; Wang, C.; Shu, C. C70-Carboxyfullerenes as Efficient Antioxidants to Protect Cells against Oxidative-Induced Stress. ACS Appl. Mater. Interfaces 2013, 5, 11101−11107. (32) Cai, X.; Hao, J.; Zhang, X.; Yu, B.; Ren, J.; Luo, C.; Li, Q.; Huang, Q.; Shi, X.; Li, W.; Liu, J. The Polyhydroxylated Fullerene Derivative C60(OH)24 Protects Mice from Ionizing-RadiationInduced Immune and Mitochondrial Dysfunction. Toxicol. Appl. Pharmacol. 2010, 243, 27−34. (33) Dugan, L. L.; Lovett, E. G.; Quick, K. L.; Lotharius, J.; Lin, T. T.; O’Malley, K. L. Fullerene-Based Antioxidants and Neurodegenerative Disorders. Parkinsonism Relat. Disord. 2001, 7, 243−246. (34) Sano, M.; Fukuda, K. Activation of Mitochondrial Biogenesis by Hormesis. Circ. Res. 2008, 103, 1191−1193. (35) Chandel, N. S.; McClintock, D. S.; Feliciano, C. E.; Wood, T. M.; Melendez, J. A.; Rodriguez, A. M.; Schumacker, P. T. Reactive Oxygen Species Generated at Mitochondrial Complex III Stabilize Hypoxia-Inducible Factor-1Alpha during Hypoxia: a Mechanism of O2 Sensing. J. Biol. Chem. 2000, 275, 25130−25138. (36) Klaunig, J. E.; Kamendulis, L. M.; Hocevar, B. A. Oxidative Stress and Oxidative Damage in Carcinogenesis. Toxicol. Pathol. 2010, 38, 96−109. (37) Santos, S. M.; Dinis, A. M.; Peixoto, F.; Ferreira, L.; Jurado, A. S.; Videira, R. A. Interaction of Fullerene Nanoparticles with Biomembranes: from the Partition in Lipid Membranes to Effects on Mitochondrial Bioenergetics. Toxicol. Sci. 2014, 138, 117−129. (38) Chen, Z.; Ma, L.; Liu, Y.; Chen, C. Applications of Functionalized Fullerenes in Tumor Theranostics. Theranostics 2012, 2, 238−250. (39) Chiu, I. M.; Touhalisky, K.; Liu, Y.; Yates, A.; Frostholm, A. Tumorigenesis in Transgenic Mice in Which the SV40 T Antigen is Driven by the Brain-Specific FGF1 Promoter. Oncogene 2000, 19, 6229−6239. (40) Hsu, Y. C.; Lee, D. C.; Chen, S. L.; Liao, W. C.; Lin, J. W.; Chiu, W. T.; Chiu, I. M. Brain-specific 1B Promoter of FGF1 Gene Facilitates the Isolation of Neural Stem/Progenitor Cells with SelfRenewal and Multipotent Capacities. Dev. Dyn. 2009, 238, 302−314. (41) Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Stages of Embryonic Development of the Zebrafish. Dev. Dyn. 1995, 203, 253−310. (42) Wu, C. C.; Tsai, T. H.; Chang, C.; Lee, T. T.; Lin, C.; Cheng, I. H.; Sun, M. C.; Chuang, Y. J.; Chen, B. S. On the Crucial Cerebellar Wound Healing-Related Pathways and Their Cross-talks after Traumatic Brain Injury in Danio rerio. PLoS One 2014, 9, e97902.

11492

DOI: 10.1021/acsami.7b01077 ACS Appl. Mater. Interfaces 2017, 9, 11482−11492