Toward Understanding the Antitumor Effects of Water-Soluble

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Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

Toward Understanding the Antitumor Effects of Water-Soluble Fullerene Derivatives on Lung Cancer Cells: Apoptosis or Autophagy Pathways? Chui-Wei Wong,† Alexander V. Zhilenkov,§ Olga A. Kraevaya,§,∥ Denis V. Mischenko,§ Pavel A. Troshin,*,§,∥ and Shan-hui Hsu*,†,‡,⊥

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†

Institute of Polymer Science and Engineering and ‡Research and Development Center for Medical Devices, National Taiwan University, Taipei 10617, Taiwan § Institute for Problems of Chemical Physics of Russian Academy of Sciences, Chernogolovka 142432, Russian Federation ∥ Skolkovo Institute of Science and Technology, Moscow 143026, Russian Federation ⊥ Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli 35053, Taiwan S Supporting Information *

ABSTRACT: Here we report the synthesis and investigation of anticancer effects of a series of water-soluble fullerene derivatives bearing amino acid (F1−F7) and thioacid (F8−F10) residues. Compounds F4 and F10 efficiently inhibited proliferation of lung cancer cells in vitro while being nontoxic to endothelial cells. It was revealed that the cancer cell death was caused by either autophagy (F4) or apoptosis (F10). Both fullerene derivatives strongly inhibited the tumor growth in the zebrafish xenograft model. In contrast to the vast majority of known cytostatics, fullerene derivatives do not show any significant acute toxicity effects in mice. Importantly, functional groups attached to the carbon cage affect interaction of the compounds with cancer cells, thus enabling realization of two different cell death mechanisms. The obtained results pave a way to the development of a new generation of selective antitumor drugs suppressing efficiently the proliferation of cancer cells while being nontoxic to normal cells.

1. INTRODUCTION

illumination, so they are intensively explored as potential drugs for photodynamic antitumor therapy.8−10 Another very promising research direction is based on combining standard clinical cytostatic drugs with the fullerene derivatives.11 In particular, this approach is very beneficial when using hydrophobic anticancer drugs such as paclitaxel.12 The chemotherapeutic drugs, such as anthracycline antibiotics, cause cancer cell death by generating excessive oxidative stress.13,14 Meanwhile, water-soluble fullerene derivatives have plenty of conjugated double bonds that have strong activity with respect to the addition of radical species and, therefore, serve as radical sponges. These radical scavenging properties mitigate the side effects of oxidative stress caused in normal cells by standard antitumor drugs and slightly enhance their therapeutic efficiency against cancer due to passive targeting effects.15 In other words, some water-soluble fullerene derivatives might exert a protective effect from the oxidative stress generated by the classical small-molecule drugs.16 Additionally, fullerenes might induce structural and elastic property changes in the lipid membrane,17 thus allowing these compounds and loaded drug molecules to enter the cells and

Implementation of nanotechnology opened broad opportunities for the development of modern medicine, especially in the field of cancer therapeutics. The unique geometric features, and physicochemical and, particularly, surface properties of nanomaterials facilitate their interactions with biological targets, making them much more efficient compared with the conventional small-molecule drugs.1 Fullerene C60, originally named buckminsterfullerene, represents a symmetrical hollow molecule with the cagelike structure composed of 20 hexagons and 12 pentagons2 and has a molecular diameter of approximately 1 nm. Fullerene C60 and its derivatives form an important family of nanomaterials2 though it has negligibly low solubility in aqueous media and undergoes aggregation very easily,3,4 which hamper its biomedical applications. Attaining high aqueous solubility for hydroxylated fullerenes C60(OH)n obtained via chemical modification of C60 in the early 1990s inspired intense exploration of the potential of water-soluble fullerenes in the field of biomedicine.5 Watersoluble fullerene derivatives are frequently tested as delivery vehicles for anticancer drugs and magnetic resonance imaging contrast agents.6,7 Fullerene derivatives also generate efficiently single oxygen and other reactive oxygen species under © XXXX American Chemical Society

Received: April 17, 2019

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DOI: 10.1021/acs.jmedchem.9b00652 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Molecular structures of the fullerene derivatives F1−F10 investigated in this work.

cross the blood−brain barrier (BBB),18,19 which can hardly be achieved for the vast majority of conventional drugs used alone. Intrinsic antitumor activity of the fullerene derivatives is rarely observed due to the generally low toxicity of these compounds. Therefore, it was reported until now only for materials with brutto-compositions C 60 (OH) 20 , 20 ,21 C60(OH)22,22 C60(OH)x,23 Gd@C82(OH)22,24−26 adduct of Gd@C82 with alanine,27 C60C(C6H3(COONa)2)2,28 Diels− Alder adduct of C60 with steroid,29 pyrrolidinium fullerene derivatives,30,31 and Pd/Pt-[60] fullerene complexes.32 It should be emphasized that the vast majority of the aforementioned compounds represent poorly characterized complicated mixtures of multiple chemical species besides very few individual compounds with almost zero solubility in aqueous systems, leading to severe problems with their excretion after administration in vivo. Our recent study highlighted a series of individual water-soluble fullerene derivatives with different surface functional groups, which produced either a neuroprotective or antiglioma effects, emphasizing the potential of C60 derivatives in treating various central nervous system-related diseases by changing the surface functional groups on the carbon cage.19 Lung cancer is the most common cause of cancer-related death in the world. The overall 5 year survival rate is lower than that of many other common cancers.33 About 85% of lung cancers belong to the nonsmall cell lung cancer (NSCLC).34 The metastasis, chemotherapeutic resistance, and recurrence are the major issues of NSCLC.33,34 To overcome these challenges, nanotechnology has been employed to deliver drugs to the lung through intravenous and oral routes. For example, albumin nanoparticles (∼130 nm) packed with

paclitaxel (poorly soluble antimitotic drug) were administered intravenously to patients to prevent the hypersensitivity induced by the solvent of paclitaxel (Cremophor) and to increase the drug uptake in tumors through the extracellular matrix glycoprotein.35 Nanoparticles could protect paclitaxel from being recognized and excreted by the P-glycoprotein efflux pump in the intestine36 and thus improve its oral bioavailability for oral chemotherapy of lung cancer.37 Different from the aforementioned approaches, a couple of more recent studies demonstrated that iron oxide or zinc oxide nanoparticles alone without drug loading induced significant cytotoxicity, apoptosis, and autophagy in cancer cells through the generation of reactive oxygen species (ROS).38,39 Apoptosis, autophagy, and necrosis are three essential programmed cell death mechanisms for organisms to maintain cell homeostasis.40 The balance in ROS generation and antioxidant function of cells leads to cellular redox homeostasis. When the ROS production is excessive or the antioxidant system capacity is impaired, the mitochondrial processes and membrane integrity are severely impacted, leading to a mitochondrial dysfunction. Therefore, ROS is a critical player in cell apoptosis. Other than the apoptotic pathway, nanomaterial-induced cellular autophagy might also be correlated with mitochondrial damage.38,41 It should be emphasized that the effects of different surface functional groups attached to the nanomaterial particles in the context of inducing either apoptosis or autophagy as major pathways for killing the cancer cells still remain unclear. In the present work, we synthesized ten different watersoluble fullerene derivatives (Figure 1) and analyzed their antitumor action in lung cancer. In vitro experiments were performed on NSCLC tumor cells to select the most promising B



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Scheme 1. Synthesis of the Fullerene Derivatives F1−F10

performed for aqueous solutions of F1−F10 confirmed their acceptably high purity (Figure S1, Supporting Information (SI)) 2.2. Aggregation Behavior of Fullerene Derivatives in Aqueous Solutions. It is well known that water-soluble fullerene derivatives readily form in aqueous media various supramolecular structures such as micelles, bi- and multilayer vesicles, and clusters of micelles/vesicles (Figure 2A).44 The aggregation behavior of the fullerene derivatives F1−F10 in aqueous solutions was investigated by dynamic light scattering (DLS), and the obtained size distribution profiles are given in Figure 2B. The smallest particles with the hydrodynamic radius ⟨Rh⟩ of 2−3 nm detected only in solution of the fullerene derivative F3 most probably represent small micelles (Type I in Figure 2A). Larger particles with ⟨Rh⟩ of 30−100 nm correspond to bilayer or multilayer vesicles (Type II and Type III, Figure 2A). The biggest particles with the hydrodynamic radius ⟨Rh⟩ of >500 nm can be considered as clusters of the smaller vesicles (Type IV, Figure 2A). The obtained DLS results confirm that water-soluble fullerene derivatives form different types of supramolecular associates in aqueous solutions, whereas the individual solvated molecules were not detected probably due to their low concentration in the system and smaller light-scattering efficiencies. It should be emphasized that supramolecular association of the water-soluble fullerene derivatives converts them to complex nanoparticles, which are expected to demonstrate different biological activities compared with truly solubilized compounds represented by individual solvated molecules. 2.3. Viability of A549 Treated with Functionalized Fullerenes. The cytotoxicity of water-soluble fullerene derivatives with respect to NSCLC cells was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the Cell Counting Kit-8 (CCK-8)

candidates. Three-dimensional NSCLC tumor spheroids were employed to examine the tumor suppression activities by the fullerene derivatives. Finally, the antitumor efficacy of C60 derivatives in vivo was assessed using the zebrafish model. Our studies provide evidence that different surface functionalizations of fullerenes might enable unique properties and distinct regulation of the mitochondrial function in target cells, resulting in either apoptosis or autophagy effect. Such switching of the predominant cell death mechanism by surface chemistry provides an impetus to the development of even more potent functionalized C60 derivatives for lung cancer treatment.

2. RESULTS 2.1. Synthesis of Water-Soluble Fullerene Derivatives. The chemical structures of the water-soluble fullerene derivatives employed in this study are displayed in Figure 1. They represent two families of compounds with different linkages between the fullerene cage and the solubilizing addends: C−N bonds (compounds F1−F7 with amino acid residues) and C−S bonds (compounds F8−F10 with thioacid residues). The fullerene derivatives F1−F10 were synthesized using previously reported general methods based on the reactions of C60Cl6 with tert-butyl esters of amino acids42 or nonprotected thioacids43 according to Scheme 1. All synthesized fullerene derivatives were characterized by high-performance liquid chromatography (HPLC, for tert-butyl esters), mass spectrometry, Fourier transform infrared (FTIR), and NMR spectroscopy. The obtained spectral data summarized in the Experimental Section match well the previously reported characteristics of similar compounds42,43 and fully support the molecular compositions and structures of compounds F1− F10. Gel permeation chromatography (GPC) analysis C



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Figure 2. Aggregation behavior of fullerene derivatives. (A) Four major types of supramolecular structures formed in aqueous solutions of fullerene derivatives. (B) DLS profiles revealing particle size distribution in aqueous solutions of the fullerene derivatives F1−F10.

Figure 3. Effects of water-soluble fullerene derivatives on the viability of lung cancer cells. (A) Viability of A549 cells treated with various functionalized fullerene derivatives for 72 h as evaluated by the MTT assay. Cell viability % = [(mean optical density of the sample − blank)/(mean optical density of the control − blank)] × 100%. Results are shown as mean ± standard error of the mean. Asterisks indicate statistically significant differences, **, p < 0.01; ****, p < 0.001. (B, C) Effects of the four selected functionalized fullerenes on the viability of various NSCLC cancer cell lines, A549, H1299, and H460 (B), and on endothelial cells (C). The cell viability was determined following the cells’ treatment with F3, F4, F9, or F10 for 72 h by the CCK-8 assay.

assay (details given in the Experimental Section). As shown in Figure 3A, compounds F1−F7 in a concentration of 200 μM induced significant cytotoxicity in A549 cells. In particular, F4 caused the most severe cell death among this group of compounds. Within the second group of compounds (F8− F10), the fullerene derivative F10 showed the highest cytotoxicity in A549 cells. The effect of the selected fullerene derivatives (F3, F4, F9, and F10) taken in different concentrations on the cell viability in three different NSCLC cancer cell lines (A549, H1299, and H460) was examined by the CCK-8 assay revealing the halflethal inhibition concentrations (IC50). The IC50 values for F3, F4, F9, and F10 were estimated to be in the range between ∼38 and ∼360 μM (Figure 3B and Table 1). These data were consistent with the results of the MTT assay, that is, F4 and F10 were more effective than F3 and F9 in the inhibition of cancer cell growth. In addition, the toxicity of fullerene derivatives with respect to normal bovine endothelial cells (BECs) was investigated, and the results are shown in Figure 3C. The viability of endothelial cells was not significantly reduced by F3, F4, or F9 in all investigated doses. Meanwhile, higher doses of F10 (≥100 μM) just slightly decreased the endothelial cell viability. We used the negative control molecule selected from previous literature data (Hsieh et al., 2017, Compound 3), which was nontoxic with respect to glioblastoma cells.19 The commercial antitumor drug cisplatin

was used as a positive control for in vitro experiments while studying the inhibitory activities of fullerene derivatives on A549 and BECs, as shown in Figure S2, Supporting Information. Taken together, these findings indicated the differential cytotoxic effects of the fullerene derivatives F4 and F10 on NSCLS cells and their low toxicity on endothelial cells. 2.4. Production of Reactive Oxygen Species (ROS) and Induction of Apoptosis in Cells by Treatment with Fullerene Derivatives. ROS production and apoptosis after treatment of cells with fullerene derivatives were, respectively, analyzed by VitaBright-48 (VB-48) and Annexin-V/7-aminoactinomycin D (7-ADD) staining (details given in the Experimental Section). The glutathione (GSH) levels represented by the VB-48 intensities for A549 cells after treatments with F4 or F10 are shown in Figure 4A. The images of the cytometric analyses revealed that the percentage of propidium iodide (PI)-negative cells with low VB-48 (i.e., D



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Table 1. IC50 Values Obtained from the Sigmoidal Fits of Dose−Response Viability Shown in Figure 3B fullerenes

IC50 value (μM)

cell lines

F3

F4

F9

F10

A549 H460 H1299

169.6 ± 1.19 360.6 ± 1.35 *a

144.9 ± 1.23 103.0 ± 1.05 38.6 ± 1.14

77.7 ± 1.20 250.3 ± 1.14 273.5 ± 1.30

93.1 ± 1.12 121.5 ± 1.16 128.8 ± 1.19

Dose−response data not amenable to sigmoidal fitting.

a

became dead cells after treatment with F10. Based on these results, F10 induced stronger intracellular ROS production and caused more severe cell death as compared with F4. The cell apoptosis state assayed by NucleoCounter NC3000 using Annexin-V and 7-ADD staining is shown in Figure 4C. The percentage of Annexin-V-positive cells increased after treatment with F4 or F10. The double-positive cells stained with Annexin-V and 7-AAD are considered to be in a late apoptotic state. As shown in Figure 4C, the percentage of the double-positive cells (late apoptosis) increased from 15% (untreated control) to 22.3% after the treatment with F10 (200 μM) for 6 h. As indicated in Figure 4D, the sums of early and late apoptotic cells also increased after the treatment with F10. Meanwhile, the values were not significantly different between the MOCK group and the group treated with F4. These results indicate that F10 induces strong apoptosis in A549 cells, whereas F4 does not show that pronounced apoptotic activity. 2.5. Reduction of Mitochondrial Functions by Fullerene Derivatives. To evaluate the mitochondrial function after treatment of the cells with selected fullerene derivatives, the values of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed as described in the Experimental Section. The OCR and ECAR values, reflecting the cell metabolism with and without fullerene addition, are displayed in Figure 5. Both the OCR and ECAR values decreased in A549 cells after treatment with F4 or F10 (Figure 5A,B). Meanwhile, the basal OCR value of A549 cells significantly decreased after treatment with F4 for 72 h (Figure 5A). Moreover, F4 induced significant decreases in the basal respiration, adenosine triphosphate (ATP) production, maximal respiration, and proton leak production (Figure 5C). The ATP production was decreased by 3.2-fold at 24 h and 7.1-fold at 72 h after addition of F4. F10 showed a much less pronounced effect on the ATP production as compared with F4. Further experiments performed for the cells stained with a mitochondria-specific immunofluorescent label (anti-hexokinase I) also confirmed the colocalization of fullerenes with the mitochondria (Figure S3, SI). Taken together, these results indicated that fullerene derivatives F4 and F10 inhibit the proliferation of A549 cells by suppressing their mitochondrial functions. 2.6. Autophagy Induced by Fullerene Derivatives. We have demonstrated that the reduction of the cell population after their treatment with F4 was not related to apoptotic cell death. We thus further investigated the effect of F4 on the autophagy of A549 cells treated with this fullerene derivative for 72 h. The obtained results are shown in Figure 6. The microtubule-associated protein 1A/1B light chain 3B (LC3B) puncta expression in A549 was clearly induced by the treatment of cells with F4 (Figure 6A, green dots). The observed LC3B accumulation indicated the increase in the autophagosome formation. The protein level of LC3B-II

Figure 4. Apoptosis induced by the water-soluble fullerene derivatives in A549 cells. (A) Scatter plots showing changes in propidium iodide (PI) and Vitabright-48 staining in response to the cell treatment with F4 or F10. (B) Quantification of the healthy (right lower panel), unhealthy (left lower panel), and dead (upper panel) phenotypes of the cells in (A). (C) Annexin-V/7-AAD analysis for F4- or F10treated A549. The fullerene derivatives were given at 0 h. The analyses were performed at the specified time points. The concentrations of F4 and F10 were 100 and 200 μM, respectively. (D) Quantification of the sum of the early apoptotic (right lower panel) and late apoptotic (right upper panel) phenotypes of the cells in (C). Asterisks indicate statistically significant differences, ***, p < 0.005; ****, p < 0.001.

reduced GSH levels) was 26% for the cells incubated with F10 (100 μM) for 0.5 h. This value was much higher than that for the untreated control (7.9% at 0 h). The cell death represented by the percentage of PI-positive cells was 12.8% at 0 h and increased to 24.4% after 5 h treatment of the cells with F10. On the other hand, the percentage of the cells with reduced GSH levels (unhealthy cells) was 7.9% at 0 h and increased to 13.1% at 0.5 h, but decreased to 3.1% at 5 h after cell treatment with F4 (200 μM). As shown in Figure 4B, the unhealthy cells E



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Figure 6. Autophagosome formation and cell-cycle G1 phase arrest induced by the fullerene derivative F4 in cultured A549 cells. (A) Induction of autophagy in A549 treated with 200 μM F4 (red) for 72 h as assessed by the immunofluorescence staining of the endogenous LC3B with the polyclonal anti-LC3B Ab (green). The red fluorescence of the fullerene derivative was imaged using an excitation filter Ex 330−380 and the emission long-pass barrier filter BM593. BF, bright-field microscopy. Scale bar represents 50 μm. Red arrowheads represent autophagosomes. (B) Immunoblots for the expression of indicated proteins (ATG 5−ATG 12 complex and LC3B) in A549 treated with F4 (200 μM) for 24, 48, and 72 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The folds of the expression levels of ATG 5− ATG 12 complex, LC3B-I, and LC3B-II (normalized to GAPDH) relative to the MOCK group are also listed. (C) Cell-cycle analysis for A549 treated with F4. Cells were harvested after 72 h, fixed, and stained with PI and analyzed for the DNA content. The cell-cycle distribution was assessed using the DNA stain. (D) The percentages of cells in sub-G1, G1, S, and G2/M phases of the cell cycle are represented by a bar diagram. It was evident that F4 treatment switched cells of the S phase to the G0/G1 phase. Asterisks indicate statistically significant differences, *, p < 0.05.

Figure 5. Effect of F4 and F10 fullerene derivatives (200 μM) on the mitochondrial function of A549. The baseline of mitochondrial respiration was determined by measuring the oxygen consumption rate (OCR), which was used as a parameter to study the mitochondrial function. The values of OCR and extracellular acidification rate (ECAR) are shown for cells treated with F4 (A) or F10 (B) for 24 and 72 h. (C) Comparison of the change in cellular bioenergetic parameters between A549 cells treated with either F4 or F10. The oxygen consumption rates of basal respiration, ATP production, maximal respiration, and proton leak were analyzed using Agilent Seahorse Wave 2.4 Software. Asterisks indicate statistically significant differences, *, p < 0.05; ***, p < 0.005; ****, p < 0.001.

2.7. Inhibition of Tumor Growth in Vitro and in Vivo by Fullerene Derivatives. The effects of fullerene derivatives on growth of tumor spheroids in vitro and on tumor expansion/metastasis in vivo in the zebrafish xenograft model are shown in Figure 7. The detailed protocols of zebrafish studies are given in the Experimental Section. The tumor spheroids were disintegrated after their treatment with F4 or F10. Such disintegration was more severe in the case of using F10, where the spheroids became small cell clusters after the treatment. In the case of using F4, the spheroids were flattened but the cells still remained clustered (Figure 7A). In zebrafish, the proliferation of the implanted A549 cells was markedly suppressed by the fullerene derivatives F4 and F10 as reflected

showed 12.4-fold increase, whereas that of Autophagy Related 5-Autophagy Related 12 (ATG 5−ATG 12) increased by a factor of 4.7 after treatment of the cells with F4 for 72 h (Figure 6B). Additionally, F4 induced cell-cycle arrest at the G0/G1 phase, increasing the fraction of the A549 cells found in the G0/G1 phase from ∼59.5 to ∼76.4% (Figure 6C,D). These results verified that the water-soluble fullerene derivative F4 promoted autophagy as well as cell-cycle arrest. F



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next section) and, therefore, can be considered as much more promising antitumor pharmaceuticals than cisplatin. 2.8. Acute Toxicity of the Fullerene Derivatives F2, F4, F5, and F10 in Mice. Three fullerene derivatives designed and studied in this work (F2, F4, F5, and F10) were analyzed with respect to their acute toxicity in mice (Table 2). It was Table 2. Acute Toxicity of the Fullerene Derivatives F2, F4, F5, and F10 in Mice compound

LD100 (mg/kg)

LD50 (mg/kg)

MTD (mg/kg)

F2 F4 F5 F10 aspirin cisplatin doxorubicin

600 800 1000 526 500 16 26

334 550 725 374 200 12 12

150 300 500 − − 8 9

found that all of these compounds have low toxicity since their lethal (LD100) and median lethal (LD50) doses were ca. 2−4 times higher than for one of the most common drugs known as aspirin. Moreover, the toxicity of the fullerene derivatives was much lower than for conventional cytostatics such as cisplatin and doxorubicine, both showing LD50 of 12 mg/kg in mice.

3. DISCUSSION Water-soluble fullerene derivatives, like other nanomaterials, have unique properties for potential biomedical applications. Many C60 fullerene derivatives were reported to have ROS scavenging, antioxidant, neuroprotective, and antitumor properties. Nevertheless, the mechanistic aspects, particularly the pathway of the fullerene-induced cell apoptosis, remained poorly understood. Moreover, fullerene-induced cell autophagy was not observed until now. These aspects were considered in detail in the present manuscript. In particular, fullerenols C60(OH)n were reported to behave as ROS scavengers, reducing the cell apoptosis in vitro.45 Excessive production of ROS is associated with the pathology of cancer and acute lung fibrosis.46 Fullerene derivatives have been investigated in numerous antineoplastic applications including fibrosis and cancer because of their ROS quenching and antioxidant properties. Water-soluble fullerenes reduced the severity of pulmonary fibrosis induced by bleomycin in mice.47 Little or no pulmonary toxicity was observed in rats after intratracheal instillation of C60 or C60(OH)24.48 Several previous studies indicated that the pristine C60 fullerene had no cytotoxic effects on cancer cells. The malignant breast epithelial cells exposed to the pristine C60 fullerene in doses of up to 200 μg/mL did not show any changes in the cell morphology, cytoskeleton organization, cell-cycle dynamics, or cell proliferation.49 The pristine C60 fullerene particles were aggregated and detected in the cytoplasm and lysosomes of A549 cells, but neither apoptosis nor necrosis was observed.50 On the other hand, C60 fullerene could act as a protective agent when combined with the anticancer drug that produced high levels of ROS such as doxorubicin.51 Based on these studies, one might conclude that fullerenes often have the antioxidant (ROS scavenging) effect rather than the ROS-inducing effect. However, some water-soluble fullerene derivatives revealed cytotoxic effects.19 Two chemical classes of water-soluble fullerene derivatives were examined in this study. They had different types of

Figure 7. Suppression of tumor spheroid formation in vitro and tumor metastasis in vivo (zebrafish) for A549 cells treated with cisplatin, F4, or F10 fullerene derivatives. (A) Time-lapse images of tumor spheroids treated with 200 μM of either F4 or F10 fullerene derivative. The fullerene derivative was introduced to the spheroids after cells were incubated for 72 h for spheroid formation. The effect on spheroid morphology was monitored for another 24 h. Scale bar represents 250 μm. (B) In the zebrafish xenograft model, the stable clones of A549-pCDH cells with green fluorescent protein (GFP) were pretreated with F4 or F10 for 72 h. The cells were injected into the yolk sac of wild-type zebrafish embryos at 4 h post fertilization. In vivo imaging was performed after 3, 6, and 9 days. Red arrowheads indicate A549-pCDH cells. (C) Relative tumor size (%) of the xenografted tumors evaluated at 3, 6, and 9 days. The asterisks in panel C indicate statistically significant differences between the MOCK group and the groups treated with 5 μM cisplatin, 200 μM F4 or F10 fullerene derivatives groups at 9 days; *, p < 0.001.

in both the decrease in tumor size and lowered metastatic frequency (Figure 7B,C). After quantification, the tumor size of zebrafish receiving the MOCK cells increased to ∼174% (relative to the 3 days size) after 9 days. Meanwhile, the tumor size for the groups receiving F4- or F10-treated cells for 9 days became significantly smaller, amounting to ∼28.8 and ∼62.2%, respectively. Thus, F4 inhibited the tumor growth more efficiently than F10. A549-pCDH treated with Cisplatin (5 μM, positive controls) and MOCK (negative control) were also injected into zebrafish, and the data are shown in Figures 7 and S4, SI. The effect induced by cisplatin was somewhat stronger but still comparable to the antitumor activities of F4 and F10. However, cisplatin is highly toxic and induces multiple side effects in patients, whereas the fullerene derivatives F4 and F10 demonstrate low toxicity (see the G



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cells even increased after the treatment with F4 (200 μM), which correlates with the reported earlier induction of vasoprotective effects by certain fullerene derivatives under the conditions of drug injury.50 Indeed, we showed that the intracellular ROS immediately produced after addition of the fullerene derivatives were eliminated after 5 h, which suggests efficient intracellular ROS-quenching activity of F4. This finding was consistent with the literature data showing that fullerene derivatives C60 (OH) 22 and C60 (C(COOH) 2 ) 2 repaired endothelial cells and inhibited actin depolymerization via attenuating the intracellular ROS.61 Recent publications demonstrate that the hydroxylated fullerenes affected the protein kinase B (AKT) and extracellular protein regulated kinases (ERK) pathways in mouse dorsal root ganglia neurons.62 The AKT pathway is one of the important pathways in endothelial cell biology, such as endothelial cell proliferation, differentiation, and migration.63 Besides, a previous report indicated that the fullerene derivatives C60(OH)22 and C60(C(COOH)2)2 protected primary rat brain cerebral microvessel endothelial cells against nitrogen oxide (NO)-induced damage.61 Therefore, the fact that viability of BECs slightly increased after their treatment with 100 μM F3, F4, or F9 can be probably associated with the radical scavenging activity of C60 derivatives, for example, quenching NO61 and removal of ROS (observed especially for F4 fullerene as shown in Figure 4A), which might result in the acceleration of the endothelial repair effect. Therefore, one can hypothesize that the fullerene derivatives protect the endothelial cells from ROS-induced damage and might activate the AKT and ERK pathways to promote cell proliferation. A previous study showed that gold nanoparticles (AuNPs ∼5 nm) were able to scavenge free radicals similar to the fullerene derivatives.64 At the same time, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) causes cancer cell death while being not toxic for normal cells. The gold nanoparticles conjugated with TRAIL might improve TRAIL-based therapy, which usually does not affect or slightly improves endothelial cell (EC) viabilities when used in high doses.65,66 Consistently, the cell viabilities were not significantly reduced by F3 or F9; meanwhile, higher doses of F4 slightly increased the endothelial cell viability (Figure 3C). Therefore, fullerene derivatives reported here behave in a similar way as TRAIL-loaded gold nanoparticles. However, there are not so many chemotherapeutic compounds based on small molecules reported in the literature that do not affect proliferation of endothelial cells. It is notable that the increase in intracellular ROS upon fullerene addition was not correlated with the amount of cellular uptake (Figure S5, SI). This was different from the previous results reported in the literature for other compounds.67 We assumed that the difference in cytotoxicity of the fullerene derivatives with respect to various types of cells might be ascribed to the different interactions of the surface functional groups in these compounds with cellular membranes and intracellular targets. Among all intracellular organelles, mitochondria are essentially important regulators of cell death.68 When A549 cells were exposed to F4, the fullerene derivative was detected in the cytoplasm; no ROS accumulation or apoptosis was observed after 5−6 h (Figure 4A,C), whereas cell proliferation was inhibited. It is known that water-soluble fullerene derivatives could cross the cell membrane and target the mitochondria.69 We thus further examined whether F4 may

linkages between the fullerene cage and the solubilizing addends: C−N bonds (compounds F1−F7) and C−S bonds (compounds F8−F10). Even the compounds belonging to the same family exerted various cytotoxic effects on A549 cells. Fullerene derivatives F4 and F10 were selected for further comparison because of their most pronounced cytotoxic effects on A549 cells. In spite of similar cytotoxic effects, F4 (C−N bonds) induced less ROS production than F10, whereas F4 was shown to induce autophagy in A549 cells via activation of the mitochondrial dysfunction. Meanwhile, F10 (C−S bonds) induced severe ROS production, reduced the mitochondrial function, and caused cell death through the apoptosis pathway. Both fullerene derivatives F4 and F10 reduced the tumor size in the zebrafish xenograft model. These observations associated with the antitumor effect of the fullerene derivatives F4 and F10 are summarized in Figure 8.

Figure 8. Fullerene derivatives with different functional groups can either induce or reduce ROS production and promote different cell death pathways, thus enabling antitumor effects in NSCLC cells. F4 significantly reduces ROS production, induces mitochondrial dysfunction, and promotes autophagy. F10 induces ROS production and destroys the mitochondrial function, leading to antitumor response.

Fullerene derivatives with attached residues of amino acids and peptides have been actively synthesized and explored by many groups worldwide.52−60 Our previous study revealed that water-soluble fullerene derivatives bearing residues of such amino acids as serine and phenylalanine promoted the anticancer effect depending on the surface functional groups attached to the carbon cage.19 The current study first examined the sensitivity of NSCLC cancer cell lines (A549, H460, and H1299) to various water-soluble fullerene derivatives. While starting this work, we used different concentrations of fullerene derivatives for the initial screening. The dose of 200 μM was found to be the most suitable concentration to select promising lead compounds in this study. An apparent difference in cytotoxic effects was observed among the compounds in three different cancer cell lines (Table 1). Meanwhile, the endothelial cell viability was just slightly reduced by F10 (∼28% compared with the untreated control in Figure 3C) probably due to excessive ROS production. At the same time, the population of the endothelial H



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target. Catechol or dioxybenzene represents a well-known PAINS functional group, which is incorporated in the structure of the active compound F4. However, one should not doubt the activities of compound F4 reported in this work. The PAINS-related false results are usually observed in highthroughput screening in vitro on cell cultures. However, in our work, we verified the anticancer activity of compound F4 on spheroids of tumor cells and also in vivo on xerographs of lung tumor grown in the zebrafish model. Moreover, we revealed the mechanistic aspects of the action of this compound and proved for the first time the autophagy-related pathway. Last, but not least, we observed clear structure−activity relationships for a series of compounds F1, F3, and F4. For the water-soluble fullerenes of the C−S linkages (F8, F9, and F10), the anticancer effects are stronger in the case of compounds with shorter (−CH2−)n spacers in the solubilizing addend (F9 more active than F8) and, particularly, when using sulfonic acid SO3H as the solubilizing group (F10). These structure−activity relationships might be used for the rational design of new series of fullerene derivatives with enhanced antitumor activity. Water-soluble fullerene derivatives have received much attention because of their strong radical scavenging activity and the ability to pass BBB.78 The present study showed that the water-soluble fullerene derivatives F4 and F10 had antitumor effects induced by activation of different cell death pathways. F4 induced autophagy through activation of mitochondrial dysfunction. F10 induced apoptosis due to ROS accumulation and reduction of the mitochondrial function. Notably, significant autophagy occurred only when the cells were subjected to F4 treatment, whereas significant apoptosis was observed only in the case of F10 treatment (Figure S7, SI). The modest antitumor effects caused by F3 and F9 were puzzling since they are not associated with the aforementioned pathways. Since lung cancer might have brain metastasis, the water-soluble fullerene derivatives could hypothetically serve as potential drugs for preventing brain metastasis of lung cancer. The IC50 values obtained in this work were relatively high (>50 μM), thus suggesting modest antitumor activity of the investigated compounds. Further molecular design might deliver more promising compounds while exploiting the initial structure−activity relationships identified in this work. At the same time, the compounds presented in this work still have a certain potential for therapeutic applications since these fullerene derivatives have very low acute toxicity in mice. For instance, the most efficient fullerene derivative F4 has a median lethal dose LD50 of 550 mg/kg in mice, which means that it is ∼45 times less toxic than cisplatin or doxorubicin (LD50 = 12 mg/kg for both these drugs). Moreover, F4 seems to be ∼3 times less toxic than the commonly used drug aspirin (LD50 = 200 mg/kg). Therefore, the fullerene derivatives can be used in significantly higher therapeutic doses than standard cytostatic drugs such as cisplatin or doxorubicin. Moreover, the results obtained in this work reveal some structure−efficiency relationships, which can be used for rational design of more potent fullerene derivatives, for example, with increased number of functional groups on the carbon cage, thus delivering a new promising approach to treat NSCLC in the future.

induce mitochondrial-associated cytoprotective antioxidant effects or antitumor effects. Our evaluation indicated that the treatment of the cells with F4 affected the oxygen metabolism (OCR). The basal respiration and ATP production were both significantly reduced by F4 in cancer cells. These findings suggested that F4 might cause the death of A549 cells through ATP depletion via mitochondrial damage, leading to the antitumor effect. In recent years, the autophagy pathway has attracted a great deal of research interest in cancer therapy.70 The silver, gold, and titanium dioxide nanoparticles were recently reported to induce autophagy,71−73 in addition to the more commonly reported ROS-induced apoptosis.74,75 The defects of autophagy are related to cell death.76 Until now, it was unclear if the water-soluble fullerene derivatives can induce autophagy or not. In the present study, we investigated the role of autophagy in A549 cells after the treatment with F4. Indeed, the cells exhibited autophagosome accumulation after the addition of F4 (Figure 6B). The degradation process of autophagosome depends on the activity of lysosomes. The literature indicated that silica nanoparticles triggered cellular autophagy dysfunction via lysosomal impairment and inhibited degradation of autophagosome in hepatocytes.77 As mentioned earlier, the pristine fullerene derivatives entered cells and aggregated in lysosomes.50 A very recent study showed that the defect of lysosomal membrane permeability could disturb the fusion of normal autophagosome with a lysosome to degrade the encapsulated content, and subsequently, the misfolded proteins and impaired organelles might accumulate in the cells, causing cell death.77 The autophagic cell death after the treatment with F4 might be due to the inhibition of autophagosome degradation. This unique pathway, however, was not found for other fullerene derivatives (including F3, F7, and F10) involved in this study (Figure S6, SI). Another interesting finding was that F4 showed less antitumor effect in vitro than F10 but had stronger antitumor effect in vivo (Figure 7). Taking into account a good endothelial cell response of F4, the autophagy mechanism revealed for this new fullerene derivative might offer significant advantage for designing the surface chemistry of more potent water-soluble fullerene derivatives for antitumor applications in the future. The functional groups incorporated in the solubilizing addends attached to the surface of the carbon cage in watersoluble fullerene derivatives might affect cell viability in different ways. For the water-soluble fullerenes with C−N linkages incorporating a benzene ring in the addends (F1, F3, F4, and F7), the anticancer effects become stronger with increase in the number of hydroxyl (−OH) substituents in that ring (the least active F1 and F7 followed by F3 and then by the most active F4). These data suggested that the presence of the phenol (hyroxybenzene) or catechol functional groups could be critical for inducing antitumor activities. We intentionally attached the DOPA (dioxyphenylalanine) residues to the fullerene cage in compound F4 because of its unique properties. Indeed, DOPA is a nontoxic substance, which participates in multiple biological processes in animals and has pronounced biological activity and therapeutic use; for example, DOPA is a common drug for treatment of Parkinson’s disease. Pan-assay interference compounds (PAINS) are chemical substances that are often false-positive in high-throughput screens since they tend to react nonspecifically with numerous biological targets rather than specifically affect one desired I



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144.11, 144.26, 144.45, 144.74, 145.15, 145.31, 145.51, 145.79, 146.23, 147.08, 147.16, 147.24, 147.81, 148.05, 148.29, 148.34, 148.44, 148.81, 148.94, 171.86 (COOtBu), 172.34(COOtBu), 172.81 (COOtBu), 172.92 (COOtBu), 173.05 (COOtBu). ESI MS: m/z = 1917 [C60(NHCH(COOtBu)CH(OH)Ph)5OH]−. FTIR (KBr pellet, ν, cm−1): 420 (W), 438 (W), 458 (W), 472 (W), 498 (W), 516 (M), 528 (M), 542 (S), 560 (M), 570 (M), 582 (M), 602 (W), 612 (W), 624 (W), 668 (W), 710 (W), 728 (W), 756 (M), 782 (W), 800 (M), 818 (S), 844 (S), 856 (S), 884 (S), 926 (W), 934 (M), 948 (M), 962 (M), 1064 (S), 1094 (VS), 1104 (VS), 1146 (S), 1198 (M), 1240 (M), 1266 (M), 1290 (M), 1314 (W), 1340 (W), 1364 (M), 1374 (M), 1388 (M), 1396 (W), 1406 (W), 1420 (M), 1436 (M), 1448 (M), 1458 (S), 1472 (M), 1490 (W), 1498 (W), 1508 (M), 1522 (M), 1540 (M), 1560 (M), 1570 (W), 1576 (M), 1618 (M), 1624 (M), 1636 (M), 1646 (M), 1654 (M), 1662 (W), 1670 (M), 1676 (W), 1684 (M), 1700 (M), 1718 (M), 1734 (M), 1750 (M), 1762 (W), 1772 (W), 1792 (W), 1800 (W), 1810 (VW), 1830 (W), 1844 (W), 1868 (W), 2342 (M), 2360 (M), 2852 (M), 2924 (S), 3030 (W), 3064 (W), 3074 (W), 3082 (W), 3134 (W), 3266 (M), 3302 (M), 3422 (S), 3482 (S), 3502 (S), 3524 (M), 3546 (M), 3568 (M), 3588 (M), 3610 (W), 3620 (W), 3630 (M), 3650 (M), 3670 (W), 3676 (W), 3690 (W), 3712 (W), 3736 (W), 3752 (W), 3770 (W), 3802 (W), 3822 (W), 3838 (W), 3854 (M), 3870 (W), 3886 (W), 3892 (W), 3902 (W), 3918 (W). Elemental analysis: C145H130ClN5O15 (Mw = 2218.1): calcd, %: C 78.52, H 5.91, N 3.16; found, %: C 78.31, H 6.10, N 3.05. 5.1.3. Compound F3 (Tyrosine − [60]Fullerene Adduct). Purity of >95% (HPLC/GPC, elemental analysis). 1H NMR for the tert-butyl ester form (500 MHz, CDCl3, δ, ppm): 0.82−1.55 (m, 90H, CH3), 2.53−3.47 (m, 10H, CH2), 3.98−4.75 (m, 5H, CH), 6.76−7.42 (m, 20H, CH). 13C NMR for the tert-butyl ester form (126 MHz, CDCl3, δ, ppm): 27.74 (CH3), 27.76 (CH3), 27.82 (CH3), 27.87 (CH3), 27.90 (CH3), 28.82 (CH3), 40.41 (CH2), 40.58 (CH2), 40.81 (CH2), 40.92 (CH2), 41.18 (CH2), 59.12 (CH), 59.52 (CH), 60.03 (CH), 60.34 (CH), 64.36 (Csp3 of the fullerene cage), 65.58 (Csp3 of the fullerene cage), 67.19 (Csp3 of the fullerene cage), 78.23 (C(CH3)3), 78.26 (C(CH3)3), 78.29 (C(CH3)3), 81.26 (C(CH3)3), 81.38 (C(CH3)3), 81.50 (C(CH3)3), 124.03, 124.07, 124.18, 124.24, 124.56, 129.96, 130.11, 130.17, 130.29, 130.34, 142.76, 143.11, 143.59, 143.68, 143.84, 144.23, 144.51, 144.59, 145.40, 145.48, 146.23, 146.95, 147.14, 147.18, 147.23, 147.84, 147.98, 148.02, 148.27, 148.42, 148.53, 148.76, 148.84, 149.24, 150.97, 151.73, 153.75, 153.87, 153.94, 153.97, 154.13, 154.22, 154.58, 154.89, 175.06 (COOtBu), 175.12 (COO t Bu), 175.19 (COO t Bu), 175.75 (COO t Bu), 175.81 (COOtBu). ESI MS: m/z = 1091 [M − Cl]2−. FTIR (KBr pellet, ν, cm−1): 472 (W), 498 (W), 516 (W), 542 (M), 560 (W), 570 (W), 582 (W), 612 (W), 668 (W), 712 (W), 728 (W), 756 (W), 782 (W), 800 (W), 824 (M), 844 (M), 884 (M), 898 (M), 924 (W), 946 (W), 962 (W), 1066 (M), 1106 (S), 1156 (VS), 1236 (S), 1366 (S), 1390 (M), 1420 (W), 1438 (W), 1456 (M), 1474 (M), 1506 (S), 1540 (W), 1560 (W), 1570 (W), 1576 (W), 1610 (W), 1636 (W), 1646 (W), 1654 (W), 1670 (W), 1676 (W), 1684 (W), 1726 (S), 2342 (W), 2362 (W), 2868 (M), 2928 (M), 2974 (S), 3028 (W), 3302 (M), 3416 (M), 3546 (W), 3568 (W), 3588 (W), 3610 (W), 3620 (W), 3630 (W), 3650 (W), 3854 (W). Elemental analysis: C145H130ClN5O15 (Mw = 2218.1): calcd, %: C 78.52, H 5.91, N 3.16; found, %: C 78.43, H 6.02, N 3.11. 5.1.4. Compound F4 (3,4-Dihydroxyphenylalanine − [60]Fullerene Adduct). Purity of >95% (HPLC/GPC, elemental analysis). 1 H NMR for the tert-butyl ester form (500 MHz, CDCl3, δ, ppm): 1.32−1.46 (m, 135H), 2.66−2.94 (m, 10H), 3.62−3.84 (m, 5H), 7.01−7.24 (m, 15H). ESI MS: m/z = 1271 [M − Cl]2−. FTIR (KBr pellet, ν, cm−1): 472 (W), 528 (W), 622 (M), 650 (M), 662 (M), 702 (S), 770 (W), 832 (S), 924 (M), 984 (S), 1008 (S), 1050 (M), 1374 (VS), 1406 (VS), 1578 (VS), 1630 (VS), 1652 (VS), 1830 (W), 1844 (M), 1868 (M), 1918 (W), 2030 (W), 2100 (W), 2304 (W), 2340 (W), 2364 (W), 2628 (M), 2730 (M), 2930 (S), 2956 (S), 3378 (S), 3650 (W), 3670 (W), 3676 (W), 3690 (W). Elemental analysis: C165H170ClN5O20 (Mw = 2578.6): calcd, %: C 76.86, H 6.65, N 2.72; found, %: C 76.51, H 6.83, N 2.62.

4. CONCLUSIONS Among the explored water-soluble C60 fullerene derivatives, F3, F4, F9, and F10 showed the most significant cytotoxic effects with respect to NSCLC cells. Importantly, they remained nontoxic to endothelial cells and showed very low acute toxicity (e.g., LD50 for F4 is 550 mg/kg in mice). The antitumor mechanisms of fullerene derivatives F4 and F10 on NSCLS cells were different. F4 induced mitochondria dysfunction and autophagy. F10 induced ROS production and apoptosis. In spite of the different mechanisms, both these compounds could inhibit tumor growth in vivo in the zebrafish xenograft model. The obtained results revealed that variation of the solubilizing surface functional groups on the fullerene cage can switch the cell death pathways, which paves a way for rational design of a new generation of water-soluble C60 fullerene derivatives with enhanced anticancer activity. 5. EXPERIMENTAL SECTION 5.1. Synthesis of the Fullerene Derivatives. Synthesis and characterization of water-soluble fullerene derivatives F2 (βalanine[60]fullerene adduct), F5 (serine[60]fullerene adduct), and F10 (2-mercaptoethane-1-sulfonic acid[60]fullerene adduct) were reported previously.19,42,79 Compounds F1, F3, F4, F6, F7, F8, and F9 were synthesized and characterized for the first time. HPLC/ GPC and elemental analysis were used to determine the purity. All synthesized final compounds had a purity of at least 95%. 5.1.1. Synthesis of Compounds F1, F3, F4, F6, and F7. Chlorofullerene C60Cl6 (200 mg, 0.214 mmol) and toluene (100 mL) were introduced into a triple-necked round-bottom flask filled with argon. The mixture was stirred at room temperature (RT) for 1− 2 h until complete dissolution of the chlorofullerene. Anhydrous potassium carbonate (ca. 1 g) and tert-butyl ester of amino acid (3phenylserine, tyrosine, 3-hydroxytyrosine, proline, or pentafluorophenylalanine, 1.286 mmol) were added, and the reaction mixture was stirred for 30 min. Dynamics of the reaction was controlled by HPLC and TLC. When starting chlorofullerene was not observed in the reaction mixture, it was filtered through a paper filter and poured at the top of the silica gel column (Acros, 40−60 μm, 60 A). Elution with toluene−acetonitrile (100:0−95:5 v/v) mixtures produced bright orange fractions of the target compounds. Obtained solutions were analyzed using HPLC and concentrated at the rotary evaporator to afford a reddish-brown residue, which was washed with acetonitrile (3 × 30 mL) and dried in air. Tert-butyl esters of F1, F3, F4, F6, and F7 were obtained in ∼75% isolated yield. Tert-butyl protecting groups were quenched by dissolving 100 mg of the corresponding fullerene derivative in 10 mL of anhydrous CH2Cl2, adding 2 mL of trifluoroacetic acid, stirring the mixture at RT for 30 min, and removing volatiles in vacuum using a rotary evaporator. The dry residues of compounds F1, F3, F4, F6, and F7 in the form of free carboxylic acids were washed with acetonitrile (3 × 30 mL) and dried in air (∼90% yield). Treatment of the acids with the stoichiometric amount of aqueous potassium carbonate, filtration of the red-brown aqueous solutions through a 0.45 μm polyethersulfone (PES) syringe filter, and freeze-drying of the filtrates produced the target watersoluble potassium salts F1, F3, F4, F6, and F7. 5.1.2. Compound F1 (2-Amino-3-hydroxy-3-phenylpropanoic Acid − [60]Fullerene Adduct). Purity of >95% (HPLC/GPC, elemental analysis). 1H NMR for the tert-butyl ester form (500 MHz, CDCl3, δ, ppm): 0.54−1.68 (m, 90H, CH3), 3.48−4.22 (m, 5H, CH), 4.30−5.01 (m, 5H, CH). 13C NMR for the tert-butyl ester form (126 MHz, CDCl3, δ, ppm): 27.18 (CH3), 27.35 (CH3), 27.51 (CH3), 27.72 (CH3), 27.80 (CH3), 27.93 (CH3), 28.81 (CH3), 66.21 (CH), 66.43 (CH), 66.65 (CH), 66.95 (CH), 67.43 (CH), 75.36 (CH), 76.35 (CH), 76.48 (CH), 76.57 (CH), 82.31 (C(CH3)3), 82.45 (C(CH3)3), 82.57 (C(CH3)3), 82.64 (C(CH3)3), 82.81 (C(CH3)3), 126.94, 127.79, 127.91, 128.23, 139.10, 139.17, 139.32, 139.58, 139.67, 142.23, 142.59, 142.73, 143.03, 143.46, 143.86, J



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tight paper filter and then through a PES syringe filter (average pore size 0.45 μm). Hydrochloric acid was added to the filtrate to produce an orange flaky precipitate. The precipitate was collected by centrifugation, washed 5 times with deionized water, and dried in vacuum at RT. The target acids F8-OH and F9-OH were obtained as red solids with the yields of 78 and 84%, respectively. Treatment of the obtained fullerene-based decacarboxylic acids with aqueous solution (10 mL) containing stoichiometric amount of potassium carbonate followed by freeze-drying produced water-soluble potassium salts F8 and F9 with a quantitative yield. 5.1.8. Compound F8 (2-(6-Mercaptohexyl)malonic Acid − [60]Fullerene Adduct). Purity of >95% (HPLC/GPC, elemental analysis). 1H NMR (500 MHz, acetone-d6:CS2, 1:1, δ, ppm): 1.31− 1.66 (m, 30H, CH2), 1.81−1.96 (m, 20H, CH2), 3.211−3.52 (m, 15H, CH2, CH), 5.31 (s, 1H). 13C NMR (126 MHz, acetone-d6:CS2, 1:1, δ, ppm): 27.20 (CH2), 27.29 (CH2), 27.33 (CH2), 27.52 (CH2), 28.18 (CH2), 33.11 (CH2), 33.26 (CH2), 33.54 (CH2), 51.15 (CH), 51.17 (CH), 51.20 (CH), 53.99 (Csp3 of the fullerene cage), 55.92 (Csp3 of the fullerene cage), 56.79 (Csp3 of the fullerene cage), 60.46 (Csp3 of the fullerene cage-H), 143.15, 143.18, 143.20, 143.25, 143.29, 143.73, 144.01, 144.04, 144.09, 144.32, 144.37, 144.73, 145.25, 145.45, 146.53, 146.75, 146.82, 147.56, 147.91, 148.07, 148.26, 148.47, 148.52, 148.64, 150.56, 151.33, 153.36, 154.48, 169.99 (COOH), 170.15 (COOH), 170.16 (COOH). ESI MS: m/z = 1815 [M − H]−. FTIR (KBr pellet, ν, cm−1): 528 (M), 542 (M), 560 (W), 608 (M),648 (W), 668 (M), 754 (W), 794 (W), 832 (W), 910 (W), 944 (W), 1044 (M), 1054 (M), 1134 (M), 1182 (M), 1198 (M), 1206 (M), 1232 (M), 1250 (M), 1284 (M), 1326 (S), 1362 (S), 1416 (S), 1474 (M), 1578 (VS), 1684 (M), 1696 (M), 1734 (W), 2344 (M), 2362 (M), 2926 (M), 2948 (M), 3386 (S), 3396 (S), 3630 (W), 3650 (W). Elemental analysis: C105H76O20S5 (Mw = 1818.0): calcd, %: C 69.37, H 4.21, S 8.82; found, %: C 69.15, H 4.34, S 8.73. 5.1.9. Compound F9 (2-(3-Mercaptopropyl)malonic Acid − [60]Fullerene Adduct). Purity of >95% (HPLC/GPC, elemental analysis). 1H NMR (500 MHz, acetone-d6:CS2, 1:1, δ, ppm): 1.80− 1.95 (m, 10H, CH2), 1.99−2.12 (m, 10H, CH2), 3.28−3.48 (m, 15H, CH2, CH), 5.34 (s, 1H). 13 C NMR (126 MHz, acetone-d6:CS2, 1:1, δ, ppm): 27.47 (CH2), 27.53 (CH2), 27.58 (CH2), 28.13 (CH2), 28.22 (CH2), 28.31 (CH2), 32.95 (CH2), 33.24 (CH2), 33.90 (CH2), 50.45 (CH), 50.60 (CH), 50.79 (CH), 54.02 (Csp3 of the fullerene cage), 55.94 (Csp3 of the fullerene cage), 56.58 (Csp3 of the fullerene cage), 60.98 (Csp3 of the fullerene cage-H), 143.21, 143.24, 143.30, 143.69, 144.03, 144.12, 144.20, 144.38, 144.40, 144.44, 144.73, 145.28, 145.38, 146.59, 146.81, 146.88, 147.62, 147.96, 148.12, 148.32, 148.53, 148.59, 148.71, 150.52, 151.28, 153.58, 154.41, 169.97 (COOH). ESI MS: m/z = 1606 [M-H]−. FTIR (KBr pellet, ν, cm−1): 528 (M), 542 (M), 610 (M), 648 (W), 668 (W), 726 (M), 1046 (S), 1200 (S), 1362 (S), 1412 (S), 1458 (S), 1490 (M), 1580 (VS), 1670 (S), 1676 (S), 1686 (S), 1700 (S), 2344 (W), 2362 (W), 2854 (S), 2926 (S), 3404 (S). Elemental analysis: C90H46O20S5 (Mw = 1607.6): calcd, %: C 67.24, H 2.88, S 9.97; found, %: C 67.11, H 2.67, S 10.03. 5.2. Dynamic Light-Scattering (DLS) Measurements. DLS measurements were performed using a Photocor Complex photoncorrelation spectrometer. All measurements were performed at 23 °C using near-infrared laser (wavelength 790 nm). Fullerene derivatives F1−F10 (4 mg) were dissolved in deionized water (4 mL), and the solutions were filtered through a PES syringe filter (average pore size 0.45 μm) to 4 mL glass vials (10 mm diameter). 5.3. Cell Culture. Human lung carcinoma cell lines (A549, H460, and H1299) were maintained in a Roswell Park Memorial Institute (RPMI, Gibco) medium. Bovine endothelial cells (ECs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with low glucose. Both RPMI and DMEM mediums contained 10% fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic (Invitrogen). All cells were incubated at 37 °C supplied by 5% carbon dioxide. Cells were subcultured twice per week. 5.4. Cell Proliferation Assay. Cell proliferation was determined by the MTT and CCK-8 assays. The MTT assay was first employed for screening. Briefly, A549 cells (1500 cells/well) were seeded in 96-

5.1.5. Compound F6 (Proline − [60]Fullerene Adduct). Purity of >95% (HPLC/GPC, elemental analysis). 1H NMR for the tert-butyl ester form (500 MHz, CDCl3, δ, ppm): 1.17−1.63 (m, 45H, CH3), 1.74−2.53 (m, 20H, CH2), 3.18−4.50 (m, 15H, CH2, CH). 13C NMR for the tert-butyl ester form (126 MHz, CDCl3, δ, ppm): 24.25 (CH2), 24.48 (CH2), 24.65 (CH2), 24.68 (CH2), 24.76 (CH2), 27.72 (CH3), 27.75 (CH3), 27.80 (CH3), 27.84 (CH3), 27.90 (CH3), 27.92 (CH3), 28.04 (CH3), 28.12 (CH3), 31.01 (CH2), 31.12 (CH2), 31.25 (CH2), 31.36 (CH2), 31.41 (CH2), 50.54 (CH2), 50.82 (CH2), 51.32 (CH2), 51.98 (CH2), 52.98 (CH2), 61.50 (CH), 61.67 (CH), 61.78 (CH), 61.98 (CH), 62.96 (CH), 69.13 (Csp3 of the fullerene cage), 69.77 (Csp3 of the fullerene cage), 71.64 (Csp3 of the fullerene cage), 72.49 (Csp3 of the fullerene cage), 73.05 (Csp3 of the fullerene cage), 80.07 (C(CH3)3), 80.14 (C(CH3)3), 80.23 (C(CH3)3), 139.67, 140.73, 142.67, 142.88, 142.97, 143.05, 143.25, 143.31, 143.45, 143.83, 143.87, 143.93, 144.04, 144.35, 144.68, 145.03, 145.27, 145.43, 145.49, 146.29, 146.32, 146.62, 146.79, 146.83, 146.84, 146.89, 146.90, 147.04, 147.07, 147.21, 147.25, 147.32, 147.42, 147.52, 147.70, 147.84, 147.87, 149.00, 149.06, 149.21, 149.31, 149.41, 149.76, 153.40, 174.31 (COOtBu), 174.39 (COOtBu), 174.65 (COOtBu), 174.81 (COOtBu). ESI MS: m/z = 1572 [M − Cl]−. FTIR (KBr pellet, ν, cm−1): 470 (W), 496 (W), 528 (M), 542 (M), 646 (M), 668 (W), 730 (S), 754 (M), 842 (S), 908 (S), 1034 (S), 1088 (VS), 1150 (VS), 1214 (S), 1248 (S), 1288 (S), 1366 (VS), 1392 (S), 1420 (S), 1456 (S), 1476 (M), 1510 (M), 1636 (S), 1732 (VS), 2362 (M), 2870 (S), 2926 (VS), 2972 (VS), 3416 (S). Elemental analysis: C105H80ClN5O10 (Mw = 1607.3): calcd, %: C 78.47, H 5.02, N 4.36; found, %: C 78.15, H 4.94, N 4.26. 5.1.6. Compound F7 (Pentafluorophenylalanine − [60]Fullerene Adduct). Purity of >95% (HPLC/GPC, elemental analysis). 1H NMR for the tert-butyl ester form (500 MHz, CDCl3, δ, ppm): 1.05−1.67 (m, 45H, CH3), 2.94−3.64 (m, 10H, CH2), 4.04−4.84 (m, 5H, CH). 13 C NMR for the tert-butyl ester form (126 MHz, CDCl3, δ, ppm): 27.29 (CH3), 27.45 (CH3), 27.54 (CH2), 27.56 (CH2), 27.61 (CH2), 27.64 (CH2), 27.77 (CH3), 27.82 (CH2), 27.86 (CH3), 28.23 (CH3), 54.16 (CH), 57.05 (CH), 57.54 (CH), 58.09 (CH), 60.02 (CH), 64.08 (Csp3 of the fullerene cage), 64.25 (Csp3 of the fullerene cage), 65.57 (Csp3 of the fullerene cage), 66.85 (Csp3 of the fullerene cage), 70.07 (Csp3 of the fullerene cage), 76.16 (Csp3 of the fullerene cage-Cl), 81.96 (C(CH 3)3 ), 82.05 (C(CH3)3), 82.34 (C(CH3)3), 82.56 (C(CH3)3), 82.81 (C(CH3)3), 111.08, 111.23, 111.42, 136.35, 136.41, 138.27, 138.45, 139.02, 139.19, 141.16, 141.50, 142.10, 142.22, 142.64, 142.71, 142.82, 143.05, 143.11, 143.48, 143.69, 143.83, 143.92, 143.93, 144.07, 144.22, 144.29, 144.34, 144.52, 144.70, 144.83, 145.06, 145.13, 145.55, 146.55, 147.05, 147.10, 147.12, 147.19, 147.21, 147.29, 147.77, 147.93, 148.04, 148.20, 148.33, 148.39, 148.44, 148.47, 148.61, 148.74, 148.87, 148.90, 148.96, 150.02, 150.57, 151.09, 152.89, 154.01, 154.02, 154.14, 172.06 (COO t Bu), 173.27 (COOtBu), 174.06 (COOtBu), 174.16 (COOtBu). ESI MS: m/z = 1107 [M − Cl-tBu]2−. FTIR (KBr pellet, ν, cm−1): 466 (W), 472 (W), 536 (W), 558 (W), 564 (W), 604 (W), 616 (W), 646 (W), 668 (W), 694 (W), 730 (M), 744 (W), 772 (W), 842 (M), 882 (W), 924 (M), 946 (S), 968 (S), 1014 (M), 1034 (M), 1080 (M), 1126 (S), 1152 (VS), 1226 (M), 1256 (M), 1276 (M), 1304 (M), 1370 (S), 1394 (M), 1428 (M), 1458 (S), 1504 (VS), 1522 (VS), 1558 (W), 1586 (W), 1618 (W), 1656 (M), 1730 (S), 2360 (W), 2644 (W),2850 (S), 2870 (S), 2924 (S), 2954 (S), 3310 (M), 3412 (M). Elemental analysis: C125H65ClF25N5O10 (Mw = 2307.3): calcd, %: C 65.07, H 2.84, N 3.04; found, %: C 64.83, H 3.02, N 2.88. 5.1.7. Synthesis of Compounds F8−F9. Chlorofullerene C60Cl6 (200 mg, 0.214 mmol) was dissolved in toluene (100 mL) under intense stirring. A solution of 2-(3-mercaptopropyl)malonic acid (304.7 mg, 1.712 mmol, in the case of F9) or 2-(6-mercaptohexyl)malonic acid (376.6 mg, 1.712 mmol, in the case of F8) and N,Ndiisopropylethylamine (220.8 mg, 1.712 mmol) in acetonitrile (10 mL) was added to the chlorofullerene solution in one portion, and the mixture was stirred further for 5 min at RT. The formed reddishbrown precipitate was separated by filtration and dried in air. The obtained solid (387 mg of F8 or 341 mg of F9) was dissolved in 10 mL of aqueous K2CO3 (74.0 mg, 0.536 mmol) and filtered through a K



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well microplates and treated with fullerene derivatives F1−F10 (200 μM) in an RPMI culture medium. Following the incubation for 72 h, the cells were treated with the tetrazolium dye (100 μL, 0.5 mg/mL, Sigma) for 1 h. The MTT formazan crystals were then completely resolubilized by adding 100% dimethylsulfoxide, and the optical density was detected at 540 nm using a UV−vis microplate reader (SpectraMax M5). The proliferation rate of different lung cancer cells treated with fullerene derivatives was further analyzed by the CCK-8 (Sigma) assay. Briefly, A549, H460, H1299, and EC cells were plated into a well of a 96-well culture plate for 24 h and incubated with several concentrations of fullerene derivatives in a culture medium for 72 h. After 72 h, the medium was switched to a culture medium with 5 μL of the CCK-8 solution and 95 μL of the culture medium. After further culturing for 30 min, the absorbance was read at 450 nm by the microplate reader. Cell viability was determined as the percent of viable cells relative to an untreated control. 5.5. Cell Vitality, Apoptosis, and Cell-Cycle Assay. The population of A549 cells was evaluated by the cell vitality assay using the NucleoCounter NC-3000 System (ChemoMetec). Cells (4 × 105 cells/well) were treated with 200 μM fullerenes for 72 h, washed with phosphate-buffered saline (PBS), and collected by trypsinization and centrifugation. Cells without the fullerene treatment were designated as a control. Cells were stained with solution 5 (ChemoMetec) according to the manufacturer’s protocol. Solution 5 contained acridine orange (as the counterstain), propidium iodide (PI, defining dead cells), and VitaBright-48 (VB-48). VB-48 was used to determinate the level of thiols, which are involved in the progression of cell death such as reduced glutathione (GSH). Stained cells were analyzed using the NucleoCounter NC-3000 system. Cell apoptosis was determined with the PE-Annexin-V Apoptosis Detection Kit I (BD bioscience) based on the manufacturer’s instruction. Cells (1 × 106) were treated with 200 μM fullerene derivatives for indicated time periods and then resuspended in 1 mL of 1× Annexin-V binding buffer. After incubation for 15 min with 5 μL of PE-Annexin and 7-ADD, 400 μL of 1× binding buffer was applied to each tube and immediately measured using the NucleoCounter NC-3000 system. The effect of fullerenes on cell division was determined by assessing the cellular DNA content using PI staining. The A549 (1 × 106) cells were incubated with 200 μM fullerene derivatives for 72 h. The harvested cells were resuspended in PBS, centrifuged (10 min, 400 g) several times, and resuspended in a fresh PBS. After the last centrifugation, the cells were fixed by slow addition of 1 mL of 70% ethanol. The samples were stored at 4 °C until analysis. Before the flow cytometric analysis, the ethanol was removed and the cells were resuspended in a stain buffer (250 μL of the staining solution with 1000 U RNAse A (Sigma-Aldrich), 10 mL of PBS, and 0.5 mL of PI (1 mg/mL, Sigma-Aldrich)) and incubated at 37 °C for 30 min. The cellular fluorescence was acquired, analyzed, and quantified by flow cytometry (FACSCalibur, Becton Dickinson). A number of 10 000 cells was collected per sample. The degree of PI staining was used to evaluate the ratio of cells in the cell cycle (low DNA content: G1/G0 phase; intermediate DNA content: S phase; and high DNA content: G2/mitosis). 5.6. Cell Metabolism. The cell mitochondrial function in the presence of selected fullerene derivatives was analyzed by a Seahorse XFp analyzer (Seahorse Bioscience), and the protocol was according to the manufacturer’s instruction. The A549 (1.8 × 104) cells were seeded and cultured in 24-well plates for 24 h. Cells were then incubated with 200 μM fullerene derivatives for the indicated time periods. After the treatment, the cells (3 × 104 cells per well) were transferred into microchambers and incubated for 4 h for cell attachment. The medium was then switched to unbuffered DMEM (DMEM added with 1 mM sodium pyruvate, 25 mM glucose, 2 mM GlutaMax, 31 mM NaCl, and pH 7.4) and incubated at 37 °C without 5% carbon dioxide for 1 h. The baseline measurements of OCR and ECAR were taken before sequential injection of mitochondrial inhibitors. The mitochondrial respiration chain inhibitors are oligomycin (ATP synthase complex inhibitor, 1 μM), FCCP (ATP

synthesis uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, 1 μM), rotenone (complex I inhibitor, 0.5 μM), and antimycin A (complex III inhibitor, 0.5 μM). The cellular OCR was measured to assess the mitochondrial respiration activity. 5.7. Analysis of Autophagy. Autophagy was analyzed by immunofluorescence staining of autophagosomes and Western blots of the associated proteins. After treatment with fullerene derivatives for 24 h, the cells were fixed for 15 min with ice-cold 4% paraformaldehyde (PFA) at 4 °C and washed three times with PBS. PFA-fixed cells were permeabilized by 0.1% Triton-X 100/PBS, and nonspecific binding sites were blocked with a 1% bovine serum albumin in 0.1% Tween 20/PBS for 1 h. To achieve a specific staining of autophagosomes, the cells were then incubated overnight at 4 °C with anti-LC3B at 1:500, followed with secondary polyclonal antibody, Alexa Fluor-488 conjugated goat antirabbit IgG (Abcam) at 1:500. Afterward, the cells were counterstained with the fluorescent nuclear stain Hoechst 33258 (Sigma-Aldrich) at 1 μg/mL and examined by fluorescence microscopy (Leica, DMIRB). For immunoblot analysis of autophagy-associated proteins, wholecell protein lysates were prepared and analyzed by Western blotting. Cells were seeded (2.5 × 105 cells per well) in a 6-well culture plate and cultured with fullerene derivatives for 24 h. After the treatment, the cells were collected and lysed in lysis buffer containing 20 mM of N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (pH 7.5), 0.1% of NP-40, 1.5 mM MgCl2, 420 mM NaCl, and protease inhibitor cocktail (Sigma). Sample proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a poly(vinylidene fluoride) (PVDF) membrane. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline and immunoblotted with antibodies such as polyclonal antiLC3B antibody (GTX127375, Genetex), polyclonal anti-ATG 12− ATG 5 (GTX113309, Genetex), and monoclonal anti-GAPDH (GTX100118, Genetex). The images were captured and quantified using a bioluminescence imaging system (UVP BioSpectrum AC Imagine System, UVP, Upland, CA). The related protein expression was quantified by the software ImageJ. 5.8. Functional Assay by Inhibition of Tumor Spheroid Formation in Vitro. To test the suppression of A549 in spheroid formation, the A549 spheroid formation assay was performed as described in a previous work.80 A549 cells (6 × 104) were seeded on a hyaluronan grafted chitosan (CS-HA)-coated well in 24-well culture plates for 72 h to generate spheroids. After 72 h, the spheroids were treated with 200 μM fullerene derivatives for 24 h. A time-lapse recording system (Real Time Cell Culture Monitoring System, Astec) was used for tracking the process of spheroid formation. 5.9. Generation of A549 Expressing GFP for in Vivo Study. To generate the A549-GFP (named A549-pCDH in this paper) stable cell lines for in vivo xenotransplantation assay, A549 cells were infected with a pCDH expression lentiviral vector containing GFP reporter and puromycin-resistant genes (pCDH-CMV-MCS-EF1GFPpuro; System Biosciences (SBI), Mountain View, CA). Lentiviruses were produced in HEK293T cells using the GFP expression lentiviral vector encased in a viral capsid encoded by three packaging plasmids (SBI). The virus supernatant was acquired at 48 h after transfection, filtered through 0.45 μm Millex PVDF Filters (Milipore), centrifuged through Amicon Ultra Centrifugal Filters at 5000 rpm for 30 min, and used for subsequent studies. A549 cells were infected with this lentivirus. After 48 h of incubation, the GFP fluorescence-stable cell clones were selected by the administration of 1 μg/mL puromycin for 14 days. 5.10. Functional Assay by Zebrafish Xenotransplantation in Vivo. Wild-type (AB strain) zebrafish were raised and maintained at 28 °C on a 10 h light/14 h dark cycle. The pairwise mating under standard conditions generated the zebrafish embryos, and the developmental stages were determined based on criteria described previously.81 Healthy embryos were selected for the zebrafish experiment. There were four sample groups for investigation, including A549-pCDH and A549-pCDH pretreated with 200 μM of the selected fullerene derivatives or 5 μM cisplatin for 72 h. The cells were injected into the cell mass of wild-type zebrafish embryos using a L



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pressure injector (IM-300, Narishige, Japan) at 4 h post fertilization in each group (n = 35−50). The zebrafish embryos were further incubated at 34 °C, and the size of the cell mass was imaged and determined using an inverted microscope (NIKON TS100, Japan) equipped with a digital camera. To evaluate the size of injected cell mass, the larva were anesthetized in 0.01% tricaine and were examined with fluorescence microscopy at 3, 6, and 9 days post fertilization. All animal experiment handing procedures were conducted in accordance with the laboratory animal committee at National Taiwan University, Taipei, Taiwan (Approval ID: B201500056). 5.11. Statistical Analysis. The results were the mean values and standard deviation of multiple samples from at least twice or more experiments to ensure reproducibility.

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ATG 5−ATG 12, Autophagy Related 5-Autophagy Related 12; LD100, lethal dose; LD50, median lethal dose

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(1) Kang, S.-g.; Zhou, G.; Yang, P.; Liu, Y.; Sun, B.; Huynh, T.; Meng, H.; Zhao, L.; Xing, G.; Chen, C.; et al. Molecular mechanism of pancreatic tumor metastasis inhibition by Gd@ C82(OH)22 and its implication for de novo design of nanomedicine. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 15431−15436. (2) Kroto, H. W.; Heath, J. R.; O’Brien, S. C.; Curl, R. F.; Smalley, R. E. C60: buckminsterfullerene. Nature 1985, 318, 162−163. (3) Ruoff, R.; Tse, D. S.; Malhotra, R.; Lorents, D. C. Solubility of fullerene (C60) in a variety of solvents. J. Phys. Chem. A 1993, 97, 3379−3383. (4) Sivaraman, N.; Dhamodaran, R.; Kaliappan, I.; Srinivasan, T.; Rao, P. V.; Mathews, C. Solubility of C60 in organic solvents. J. Org. Chem. 1992, 57, 6077−6079. (5) Naim, A.; Shevlin, P. B. Reversible addition of hydroxide to the fullerenes. Tetrahedron Lett. 1992, 33, 7097−7100. (6) Montellano, A.; Da Ros, T.; Bianco, A.; Prato, M. Fullerene C60 as a multifunctional system for drug and gene delivery. Nanoscale 2011, 3, 4035−4041. (7) Chen, Z.; Mao, R.; Liu, Y. Fullerenes for cancer diagnosis and therapy: preparation, biological and clinical perspectives. Curr. Drug Metab. 2012, 13, 1035−1045. (8) Mroz, P.; Tegos, G. P.; Gali, H.; Wharton, T.; Sarna, T.; Hamblin, M. R. Photodynamic therapy with fullerenes. Photochem. Photobiol. Sci. 2007, 6, 1139−1149. (9) Constantin, C.; Neagu, M.; Ion, R.-M.; Gherghiceanu, M.; Stavaru, C. Fullerene−porphyrin nanostructures in photodynamic therapy. Nanomedicine 2010, 5, 307−317. (10) Rybkin, A. Y.; Belik, A. Y.; Kraevaya, O.; Khakina, E.; Zhilenkov, A.; Goryachev, N.; Volyniuk, D.; Grazulevicius, J.; Troshin, P.; Kotelnikov, A. Covalently linked water-soluble fullerene−fluorescein dyads as highly efficient photosensitizers: synthesis, photophysical properties and photochemical action. Dyes Pigm. 2019, 160, 457−466. (11) Partha, R.; Conyers, J. L. Biomedical applications of functionalized fullerene-based nanomaterials. Int. J. Nanomed. 2009, 4, 261−275. (12) Zakharian, T. Y.; Seryshev, A.; Sitharaman, B.; Gilbert, B. E.; Knight, V.; Wilson, L. J. A. Fullerene− paclitaxel chemotherapeutic: synthesis, characterization, and study of biological activity in tissue culture. J. Am. Chem. Soc. 2005, 127, 12508−12509. (13) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56, 185−229. (14) Wang, X.; Wang, W.; Li, L.; Perry, G.; Lee, H.-g.; Zhu, X. Oxidative stress and mitochondrial dysfunction in Alzheimer’s disease. Biochim. Biophys. Acta, Mol. Basis Dis. 2014, 1842, 1240−1247. (15) Zhou, Y.; Li, J.; Ma, H.; Zhen, M.; Guo, J.; Wang, L.; Jiang, L.; Shu, C.; Wang, C. Biocompatible [60]/[70] fullerenols: potent defense against oxidative injury induced by reduplicative chemotherapy. ACS Appl. Mater. Interfaces 2017, 9, 35539−35547. (16) Grebowski, J.; Kazmierska, P.; Krokosz, A. Fullerenols as a new therapeutic approach in nanomedicine. Biomed. Res. Int. 2013, 2013, 1−9. (17) Wong-Ekkabut, J.; Baoukina, S.; Triampo, W.; Tang, I.-M.; Tieleman, D. P.; Monticelli, L. Computer simulation study of fullerene translocation through lipid membranes. Nat. Nanotechnol. 2008, 3, 363−368. (18) Oberdörster, G.; Sharp, Z.; Atudorei, V.; Elder, A.; Gelein, R.; Kreyling, W.; Cox, C. Translocation of inhaled ultrafine particles to the brain. Inhalation Toxicol. 2004, 16, 437−445. (19) Hsieh, F.-Y.; Zhilenkov, A.; Voronov, I.; Khakina, E.; Mischenko, D.; Troshin, P. A.; Hsu, S.-h. Water-soluble fullerene derivatives as brain medicine: Surface chemistry determines if they are

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00652. GPC chromatograms of the fullerene derivatives; viability of cells treated with functionalized fullerenes and cisplatin; localization of four selected functionalized fullerene derivatives; suppression of tumor metastasis in vivo; cellular uptake of different fullerene derivatives; characterization of autophagosome formation induced by different fullerene derivatives and of A549 apoptosis induced by the four selected functionalized fullerenes (PDF) Molecular formula string (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +7 496522-1418. Fax: +7 496515-5420 (P.A.T.). *E-mail: [email protected]. Phone: +886-2-3366-5313. Fax: +886-2-3366-5237 (S.-h.H.). ORCID

Pavel A. Troshin: 0000-0001-9957-4140 Shan-hui Hsu: 0000-0003-3399-055X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the bilateral Taiwanese-Russian research project (MOST 105-2923- E002-003-MY3; RFBR No. 16-53-52030) and Ministry for Science and Education of the Russian Federation (Project No. 0089-2019-0010). We thank the Taiwan Zebrafish Core Facility at National Taiwan University (NTU-105-EL-00152) for the facility and technical supports.

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ABBREVIATIONS CCK-8, Cell Counting Kit-8; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide); IC50, half-lethal inhibition concentrations; BECs, bovine endothelial cells; BBB, blood−brain barrier; NSCLC, nonsmall cell lung cancer; ROS, reactive oxygen species; HPLC, high-performance liquid chromatography; GPC, gel permeation chromatography; 7ADD, Annexin-V/7-aminoactinomycin D; VB-48, VitaBright48; GSH, glutathione; PI, propidium iodide; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; LC3B, microtubule-associated proteins 1A/1B light chain 3B; M



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neuroprotective and antitumor. ACS Appl. Mater. Interfaces 2017, 9, 11482−11492. (20) Jiao, F.; Liu, Y.; Qu, Y.; Li, W.; Zhou, G.; Ge, C.; Li, Y.; Sun, B.; Chen, C. Studies on anti-tumor and antimetastatic activities of fullerenol in a mouse breast cancer model. Carbon 2010, 48, 2231− 2243. (21) Liu, Y.; Jiao, F.; Qiu, Y.; Li, W.; Qu, Y.; Tian, C.; Li, Y.; Bai, R.; Lao, F.; Zhao, Y.; et al. Immunostimulatory properties and enhanced TNF-α mediated cellular immunity for tumor therapy by C60(OH)20 nanoparticles. Nanotechnology 2009, 20, No. 415102. (22) Nie, X.; Tang, J.; Liu, Y.; Cai, R.; Miao, Q.; Zhao, Y.; Chen, C. Fullerenol inhibits the cross-talk between bone marrow-derived mesenchymal stem cells and tumor cells by regulating MAPK signaling. Nanomed. Nanotechnol. Biol. Med. 2017, 13, 1879−1890. (23) Zhu, J.; Ji, Z.; Wang, J.; Sun, R.; Zhang, X.; Gao, Y.; Sun, H.; Liu, Y.; Wang, Z.; Li, A.; et al. Tumor-inhibitory effect and immunomodulatory activity of fullerol C60(OH)x. Small 2008, 4, 1168−1175. (24) Chen, C.; Xing, G.; Wang, J.; Zhao, Y.; Li, B.; Tang, J.; Jia, G.; Wang, T.; Sun, J.; Xing, L.; et al. Multihydroxylated [Gd@C82 (OH)22]n nanoparticles: antineoplastic activity of high efficiency and low toxicity. Nano Lett. 2005, 5, 2050−2057. (25) Meng, H.; Xing, G.; Sun, B.; Zhao, F.; Lei, H.; Li, W.; Song, Y.; Chen, Z.; Yuan, H.; Wang, X.; et al. Potent angiogenesis inhibition by the particulate form of fullerene derivatives. ACS Nano 2010, 4, 2773−2783. (26) Meng, J.; Liang, X.; Chen, X.; Zhao, Y. Biological characterizations of [Gd@ C82 (OH)22]n nanoparticles as fullerene derivatives for cancer therapy. Integr. Biol. 2013, 5, 43−47. (27) Zhou, Y.; Deng, R.; Zhen, M.; Li, J.; Guan, M.; Jia, W.; Li, X.; Zhang, Y.; Yu, T.; Zou, T.; et al. Amino acid functionalized gadofullerene nanoparticles with superior antitumor activity via destruction of tumor vasculature in vivo. Biomaterials 2017, 133, 107−118. (28) Kornev, A. B.; Peregudov, A. S.; Martynenko, V. M.; Guseva, G. V.; Sashenkova, T. E.; Rybkin, A. Y.; Faingold, I. I.; Mishchenko, D. V.; Kotelnikova, R. A.; Konovalova, N. P.; et al. Synthesis and biological activity of a novel water-soluble methano [60] fullerene tetracarboxylic derivative. Mendeleev Commun. 2013, 23, 323−325. (29) Li, L.-S.; Hu, Y.-J.; Wu, Y.; Wu, Y.-L.; Yue, J.; Yang, F. Steroidfullerene adducts from Diels−Alder reactions: characterization and the effect on the activity of Ca2+-ATPase. J. Chem. Soc., Perkin Trans. 1 2001, 617−621. (30) Mashino, T.; Nishikawa, D.; Takahashi, K.; Usui, N.; Yamori, T.; Seki, M.; Endo, T.; Mochizuki, M. Antibacterial and antiproliferative activity of cationic fullerene derivatives. Bioorg. Med. Chem. Lett. 2003, 13, 4395−4397. (31) Watanabe, T.; Nakamura, S.; Ono, T.; Ui, S.; Yagi, S.; Kagawa, H.; Watanabe, H.; Ohe, T.; Mashino, T.; Fujimuro, M. Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma. Biochem. Biophys. Res. Commun. 2014, 451, 93−100. (32) Sabounchei, S. J.; Sayadi, M.; Hashemi, A.; Salehzadeh, S.; Maleki, F.; Nematollahi, D.; Mokhtari, B.; Hosseinzadeh, L. New Pd/ Pt-[60] fullerene complexes of phosphorus ylides as anticancer agents: cytotoxic investigation and DFT calculations. J. Organomet. Chem. 2018, 860, 49−58. (33) Ettinger, D. S.; Wood, D. E.; Akerley, W.; Bazhenova, L. A.; Borghaei, H.; Camidge, D. R.; Cheney, R. T.; Chirieac, L. R.; D’Amico, T. A.; Demmy, T. L.; Dilling, T. J.; Dobelbower, M. C.; Govindan, R.; Grannis, F. W., Jr.; Horn, L.; Jahan, T. M.; Komaki, R.; Krug, L. M.; Lackner, R. P.; Lanuti, M.; Lilenbaum, R.; Lin, J.; Loo, B. W., Jr.; Martins, R.; Otterson, G. A.; Patel, J. D.; Pisters, K. M.; Reckamp, K.; Riely, G. J.; Rohren, E.; Schild, S. E.; Shapiro, T. A.; Swanson, S. J.; Tauer, K.; Yang, S. C.; Gregory, K.; Hughes, M. Nonsmall cell lung cancer, version 6. 2015. J. Natl. Compr. Cancer Network 2015, 13, 515−524.

(34) Molina, J. R.; Yang, P.; Cassivi, S. D.; Schild, S. E.; Adjei, A. A. Non-small Cell Lung Cancer: Epidemiology, Risk Factors, Treatment, and Survivorship; Mayo Clinic Proceedings, Elsevier: 2008; pp 584−594. (35) Gradishar, W. J. Albumin-bound paclitaxel: a next-generation taxane. Expert Opin. Pharmacother. 2006, 7, 1041−1053. (36) Sparreboom, A.; van Asperen, J.; Mayer, U.; Schinkel, A. H.; Smit, J. W.; Meijer, D. K.; Borst, P.; Nooijen, W. J.; Beijnen, J. H.; van Tellingen, O. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2031−2045. (37) Jiang, L.; Li, X.; Liu, L.; Zhang, Q. Thiolated chitosan-modified PLA-PCL-TPGS nanoparticles for oral chemotherapy of lung cancer. Nanoscale Res. Lett. 2013, 8, 66. (38) Khan, M. I.; Mohammad, A.; Patil, G.; Naqvi, S.; Chauhan, L.; Ahmad, I. Induction of ROS, mitochondrial damage and autophagy in lung epithelial cancer cells by iron oxide nanoparticles. Biomaterials 2012, 33, 1477−1488. (39) Bai, D.-P.; Zhang, X.-F.; et al. Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells. Int. J. Nanomed. 2017, 12, 6521−6535. (40) Ouyang, L.; Shi, Z.; Zhao, S.; Wang, F. T.; Zhou, T. T.; Liu, B.; Bao, J. K. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Proliferation 2012, 45, 487−498. (41) Eidi, H.; Joubert, O.; Nemos, C.; Grandemange, S.; Mograbi, B.; Foliguet, B.; Tournebize, J.; Maincent, P.; Le Faou, A.; Aboukhamis, I.; Rihn, B. H. Drug delivery by polymeric nanoparticles induces autophagy in macrophages. Int. J. Pharm. 2012, 422, 495− 503. (42) 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. (43) 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. (44) 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. (45) Yin, J.-J.; Lao, F.; Fu, P. P.; Wamer, W. G.; Zhao, Y.; Wang, P. C.; Qiu, Y.; Sun, B.; Xing, G.; Dong, J.; et al. The scavenging of reactive oxygen species and the potential for cell protection by functionalized fullerene materials. Biomaterials 2009, 30, 611−621. (46) Schapira, R. M.; Ghio, A. J.; Effros, R. M.; Morrisey, J.; Dawson, C. A.; Hacker, A. D. Hydroxyl radicals are formed in the rat lung after asbestos instillation in vivo. Am. J. Respir. Cell Mol. Biol. 1994, 10, 573−579. (47) Dong, R.; Liu, M.; Huang, X. X.; Liu, Z.; Jiang, D. Y.; Xiao, H. J.; Dai, H. P. Effect of water-soluble C(60) fullerenes on pulmonary fibrosis induced by bleomycin in mice. Zhonghua Yi Xue Za Zhi 2017, 97, 1740−1744. (48) Sayes, C. M.; Marchione, A. A.; Reed, K. L.; Warheit, D. B. Comparative pulmonary toxicity assessments of C60 water suspensions in rats: few differences in fullerene toxicity in vivo in contrast to in vitro profiles. Nano Lett. 2007, 7, 2399−2406. (49) Levi, N.; Hantgan, R. R.; Lively, M. O.; Carroll, D. L.; Prasad, G. L. C60-fullerenes: detection of intracellular photoluminescence and lack of cytotoxic effects. J. Nanotechnol. 2006, 4, 14. (50) Horie, M.; Nishio, K.; Kato, H.; Shinohara, N.; Nakamura, A.; Fujita, K.; Kinugasa, S.; Endoh, S.; Yamamoto, K.; Yamamoto, O.; Niki, E.; Yoshida, Y.; Iwahashi, H. In vitro evaluation of cellular responses induced by stable fullerene C60 medium dispersion. J. Biochem. 2010, 148, 289−298. (51) Prylutska, S.; Grynyuk, I.; Matyshevska, O.; Prylutskyy, Y.; Evstigneev, M.; Scharff, P.; Ritter, U. C60 fullerene as synergistic N



Article

agent in tumor-inhibitory doxorubicin treatment. Drugs R&D 2014, 14, 333−340. (52) Jennepalli, S.; Pyne, S. G.; Keller, P. A. [60] Fullerenyl amino acids and peptides: a review of their synthesis and applications. RSC Adv. 2014, 4, 46383−46398. (53) Maggini, M.; Scorrano, G.; Bianco, A.; Toniolo, C.; Sijbesma, R. P.; Wudl, F.; Prato, M. Addition reactions of C60 leading to fulleroprolines. J. Chem. Soc., Chem. Commun. 1994, 3, 305−306. (54) Bianco, A.; Maggini, M.; Scorrano, G.; Toniolo, C.; Marconi, G.; Villani, C.; Prato, M. Synthesis, chiroptical properties, and configurational assignment of fulleroproline derivatives and peptides. J. Am. Chem. Soc. 1996, 118, 4072−4080. (55) Burley, G. A.; Keller, P. A.; Pyne, S. G.; Ball, G. E. Synthesis and characterization of mono-and bis-methano [60] fullerenyl amino acid derivatives and their reductive ring-opening retro-Bingel reactions. J. Org. Chem. 2002, 67, 8316−8330. (56) Wang, G.-W.; Li, J.-X.; Li, Y.-J.; Liu, Y.-C. Novel reactions of [60] fullerene with amino acid esters and carbon disulfide. J. Org. Chem. 2006, 71, 680−684. (57) Bjelaković, M.; Todorović, N.; Milić, D. An approach to nanobioparticles−synthesis and characterization of fulleropeptides. Eur. J. Org. Chem. 2012, 2012, 5291−5300. (58) Strom, T. A.; Durdagi, S.; Ersoz, S. S.; Salmas, R. E.; Supuran, C. T.; Barron, A. R. Fullerene-based inhibitors of HIV-1 protease. J. Pept. Sci. 2015, 21, 862−870. (59) Durdagi, S.; Supuran, C. T.; Strom, T. A.; Doostdar, N.; Kumar, M. K.; Barron, A. R.; Mavromoustakos, T.; Papadopoulos, M. G. In silico drug screening approach for the design of magic bullets: a successful example with anti-HIV fullerene derivatized amino acids. J. Chem. Inf. Model. 2009, 49, 1139−1143. (60) Innocenti, A.; Durdagi, S.; Doostdar, N.; Strom, T. A.; Barron, A. R.; Supuran, C. T. Nanoscale enzyme inhibitors: fullerenes inhibit carbonic anhydrase by occluding the active site entrance. Bioorg. Med. Chem. 2010, 18, 2822−2828. (61) Lao, F.; Li, W.; Han, D.; Qu, Y.; Liu, Y.; Zhao, Y.; Chen, C. Fullerene derivatives protect endothelial cells against NO-induced damage. Nanotechnology 2009, 20, No. 225103. (62) Xiao, L.; Hong, K.; Roberson, C.; Ding, M.; Fernandez, A.; Shen, F.; Jin, L.; Sonkusare, S.; Li, X. Hydroxylated fullerene: a stellar nanomedicine to treat lumbar radiculopathy via antagonizing TNF-αinduced ion channel activation, calcium signaling, and neuropeptide production. ACS Biomater. Sci. Eng. 2018, 4, 266−277. (63) Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 2000, 6, 389−395. (64) Ho, T.-T.; Lin, Y.-C.; Hsu, S.-h. Human endothelial cell response to polyurethane−gold nanocomposites. Gold Bull. 2012, 45, 161−170. (65) Huang, Y.-J.; Hsu, S.-h. TRAIL-functionalized gold nanoparticles selectively trigger apoptosis in polarized macrophages. Nanotheranostics 2017, 1, 326−337. (66) Secchiero, P.; Gonelli, A.; Carnevale, E.; Milani, D.; Pandolfi, A.; Zella, D.; Zauli, G. TRAIL promotes the survival and proliferation of primary human vascular endothelial cells by activating the Akt and ERK pathways. Circulation 2003, 107, 2250−2256. (67) Tseng, T.-C.; Hsieh, F.-Y.; Hsu, S.-h. Increased cell survival of cells exposed to superparamagnetic iron oxide nanoparticles through biomaterial substrate-induced autophagy. Biomater. Sci. 2016, 4, 670− 677. (68) Kubli, D. A.; Gustafsson, Å. B. Mitochondria and mitophagy. Circulation Research 2012, 111, 1208−1221. (69) Foley, S.; Crowley, C.; Smaihi, M.; Bonfils, C.; Erlanger, B. F.; Seta, P.; Larroque, C. Cellular localisation of a water-soluble fullerene derivative. Biochem. Biophys. Res. Commun. 2002, 294, 116−119. (70) He, S.; Li, Q.; Jiang, X.; Lu, X.; Feng, F.; Qu, W.; Chen, Y.; Sun, H. Design of small molecule autophagy modulators: a promising druggable strategy. J. Med. Chem. 2018, 61, 4656−4687. (71) Ma, X.; Wu, Y.; Jin, S.; Tian, Y.; Zhang, X.; Zhao, Y.; Yu, L.; Liang, X. J. Gold nanoparticles induce autophagosome accumulation

through size-dependent nanoparticle uptake and lysosome impairment. ACS Nano 2011, 5, 8629−8639. (72) Zhao, Y.; Howe, J. L.; Yu, Z.; Leong, D. T.; Chu, J. J.; Loo, J. S.; Ng, K. W. Exposure to titanium dioxide nanoparticles induces autophagy in primary human keratinocytes. Small 2013, 9, 387−392. (73) Mishra, A. R.; Zheng, J.; Tang, X.; Goering, P. L. Silver nanoparticle-induced autophagic-lysosomal disruption and NLRP3inflammasome activation in HepG2 cells is size-dependent. Toxicol. Sci. 2016, 150, 473−487. (74) Foldbjerg, R.; Olesen, P.; Hougaard, M.; Dang, D. A.; Hoffmann, H. J.; Autrup, H. PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes. Toxicol. Lett. 2009, 190, 156−162. (75) Gopinath, P.; Gogoi, S. K.; Sanpui, P.; Paul, A.; Chattopadhyay, A.; Ghosh, S. S. Signaling gene cascade in silver nanoparticle induced apoptosis. Colloids Surf., B 2010, 77, 240−245. (76) Tsujimoto, Y.; Shimizu, S. Another way to die: autophagic programmed cell death. Cell Death Differ. 2005, 12, 1528−1534. (77) Wang, J.; Yu, Y.; Lu, K.; Yang, M.; Li, Y.; Zhou, X.; Sun, Z. Silica nanoparticles induce autophagy dysfunction via lysosomal impairment and inhibition of autophagosome degradation in hepatocytes. Int. J. Nanomed. 2017, 12, 809−825. (78) Schuhmann, M. K.; Fluri, F. Effects of fullerenols on mouse brain microvascular endothelial cells. Int. J. Mol. Sci. 2017, 18, 1783. (79) Khakina, E. A.; Troshin, P. A. Halogenated fullerenes as precursors for the synthesis of functional derivatives of C60 and C70. Russ. Chem. Rev. 2017, 86, 805−830. (80) Huang, Y.-J.; Hsu, S.-h. Acquisition of epithelial−mesenchymal transition and cancer stem-like phenotypes within chitosan-hyaluronan membrane-derived 3D tumor spheroids. Biomaterials 2014, 35, 10070−10079. (81) 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.

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