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Photodynamic anticancer activities of water-soluble C60 derivatives and their biological consequences in a HeLa cell line
Chemico-Biological Interactions 195 (2012) 86–94
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Photodynamic anticancer activities of water-soluble C60 derivatives and their biological consequences in a HeLa cell line Zhen Hu a,⇑, Chunhua Zhang a, Yudong Huang a,⇑, Shaofan Sun a, Wenchao Guan b, Yuhuan Yao a a b
School of Chemical Engineering and Technology, Harbin Institute of Technology, Harbin 150001, China College of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e
i n f o
Article history: Received 29 August 2011 Received in revised form 10 October 2011 Accepted 7 November 2011 Available online 15 November 2011 Keywords: Fullerene Photosensitization Free radical Antitumor Apoptosis
a b s t r a c t Photodynamic therapy is an emerging, externally activatable, treatment modality for various diseases, especially for cancer therapy. The photodynamic activities of tumor targeting water-soluble C60 derivatives (WSFD) were evaluated on HeLa cells. To overcome the poor solubility, biocompatibility and selectivity of C60, we modified C60 with L-phenylalanine, folic acid and L-arginine. Consistent with their photodynamic abilities, WSFD generated the reactive oxygen species after irradiation both in water and in vitro. No dark cytotoxicity was observed using 5 lg/mL WSFD during long incubation time. Furthermore, the uptake of WSFD into HeLa cells was much more than normal cells, which indicated the WSFD had selectivity to tumor cells. Investigation of the possible photodynamic activities of WSFD demonstrated that they expressed photokilling activities by raising the level of 1O2/O2 under visible light irradiation. In parallel, following exposure of cells to WSFD and irradiation, a marked decrease in mitochondrial membrane potential, cell viability, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased malondialdehyde (MDA) production were observed. Moreover, WSFD caused significant elevation in caspase-3 activity, and induced apoptotic death. Experiments demonstrated that both chemical properties, such as the chemical structure of adduct and addend numbers, and physical properties, such as degree of aggregation, influenced the ROS-generation abilities, cellular uptake and photodynamic activities of WSFD. The results suggest that WSFD have the potential application in cancer cell inactivation by photodynamic therapy. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Photodynamic therapy (PDT) is an emerging, externally activatable, treatment modality for various diseases. The use of PDT as a cancer therapy is particularly attractive because of its fundamental specificity and selectivity [1–4]. This is due to the fact that the photosensitizer (PS) concentrates specifically within the malignant tissue so when the light is directly focused on the lesion, it causes reactive oxygen species (ROS) to be generated resulting in cellular destruction at the region of interest. For this reason, in recent years, PDT has become the subject of intense investigation as a possible treatment modality for various forms of cancer. Similar to chemotherapy, PDT still requires agents which exhibit selectivity for the target cells. Similar to radiotherapy, the mode of action with PDT involves the use of electromagnetic radiation in order to generate radical species in situ. However, PDT is a much milder approach for cancer treatment than either [5]. The reason for this lies ⇑ Corresponding authors. Tel.: +86 451 86413711; fax: +86 451 86402403 (Z. Hu). E-mail addresses: (Y. Huang).
[email protected] (Z. Hu),
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0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.11.003
in the combination of the mode of action of the PS employed and its activation in situ by relatively long wavelength, visible light. Because of their unique physicochemical properties, fullerenes (C60) are potential candidates for many biomedical applications, including PDT [6,7]. The anticancer properties of various fullerene preparations usually result from their ability to generate cytotoxic ROS when photoexcited, which is the main principle of PDT [8,9]. Various fullerenes, including pristine C60 as well as functionalized derivatives, have been previously used to carry out PDT reactions leading to cleavage of DNA strands [10,11], photoinactivation of viruses [12,13], production of oxidative damage to lipids in microsomal membranes [14], PDT-induced killing of mammalian cells in tissue culture [15,16], and even a report of regressions after PDT in a mouse tumor model [17]. Despite their potential, most C60 derivatives used in PDT have some limitations. Mainly, they are hydrophobic or have limited water solubility which leads to the modification of their optical properties and the decrease of singlet oxygen production. Although recently a lot of work has focused on developing several new strategies to improve the solubility of C60 derivatives, including modification of the fullerenes with watersoluble substituents [18–20] and solubilization using polymers
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Fig. 1. The chemical structures of (A) PFD, (B) FFD and (C) AFD.
[21], lipid membranes [22], cyclodextrin [23], calixarenes [24], the lack of specific target and the dark cytotoxicity is still a principal challenge for C60 derivatives. In order to solve the problem described above, C60 was modified by L-phenylalanine, folic acid and L-arginine in this work. L-Phenylalanine and L-arginine are necessary amino acids for human body, which have been widely used in pharmaceutical and food additive industry. It has been reported that L-phenylalanine can be used as carrier for anticancer drugs, which delivers the drug molecules into the tumor area directly [25,26]. Its transfer effect is 3–5 times higher than other amino acids. Preliminary evidence surrounding the use of folic acid seems promising for decreasing the risk of breast, cervical, pancreatic and gastrointestinal cancer. Furthermore, the discovery of high-levels expression of folic acid receptor on many human cancer cells has rendered the folic acid as an attractive candidate for development of tumor specific therapeutics [27–29]. Thus, the modifiers we chose in this work possessed good biocompatibility and targeting. The opportunity of combining C60 with Lphenylalanine, folic acid and L-arginine appears as a desirable way to develop novel PS for PDT (Fig. 1). As designed, the PS will have good selectivity to tumor cells without dark cytotoxicity. In our previous study, we have reported that water-soluble C60 derivatives could play important role in protecting cells from apoptosis induced by hydrogen peroxide and nitric oxide [30,31]. On the other hand, illumination of C60 with visible or UV light fosters its transition to a long-lived triplet excited state and the subsequent energy transfer to molecular oxygen, yielding ROS. This dual property of C60 to either quench or generate cell-damaging ROS could be therefore exploited for its development as a cytoprotective or cytotoxic anticancer agent [6]. In this work, we have synthesized three water-soluble C60 derivatives (WSFD) by nucleophilic addition reaction. The compounds were characterized by Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), liquid chromatography–mass spectrometry (LC–MS), elemental analysis and dynamic light scattering (DLS). The photodynamic effects were evaluated on HeLa human cervix uteri tumor-derived cell line. The ROS production ability of WSFD was analyzed both in cell free condition and in vitro. After incubation with WSFD and visible light irradiation the mechanisms of cellular death induced by the derivatives were studied. 2. Experimental
USA). 2,7-Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probe Inc. (Eugene, OR, USA). The reagent kits for the measurement of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ApoAlert caspase fluorescent assay kit was purchased from BD Bioscience Clontech (Palo Alto, CA, USA). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from Biosea BCL (Beijing, China). L-Phenylalanine, folic acid and L-arginine were products of Sinoreagent Co. Ltd. (Shanghai, China). 2.2. The synthesis and characterization of WSFD WSFD was synthesized by nucleophilic addition reaction. L-Phenylalanine, folic acid or L-arginine (10 mmol) and sodium hydroxide (20 mmol) were dissolved in 3 mL water, and then 30 mL ethanol was added, the resulting solution was added to a C60 toluene solution (0.1 mmol, 60 mL) dropwise, then 5 drops of 10% tetrabutylammonium hydroxide was added under stirring. The solution was stirred at room temperature under nitrogen atmosphere. To make sure the reaction was completed, the solution was stirred for 48 h. The aqueous layer was separated from the organic layer, filtered, diluted with 3 mL water. Ethanol 40 mL was then added to cause the precipitation of the product, which was further reprecipitated with H2O/ EtOH for several times. Then the product was further purified by gel exclusion chromatography using a dextran (G-25, Pharmacia biotech) column with H2O. The product was eluted first, and then unreacted L-phenylalanine, folic acid or L-arginine was eluted. The resulting solution was dried under vacuum to give the product of L-phenylalanine C60 derivatives (PFD), folic acid C60 derivatives (FFD) or L-arginine C60 derivatives (AFD) (82.7%, 75.3% and 83.6% based on converted C60, respectively). The chemical structures of WSFD were characterized by FT-IR, 1 H NMR, 13C NMR, LC–MS and elemental analysis. The aggregation properties of WSFD were characterized by DLS. The preparation of WSFD vesicles were done by dissolving WSFD (100 mg) in H2O (100 mL) to form a solution of 1 mg/mL in concentration with ultrasonication for 2 h, followed by centrifugation and decantation. Light scattering experiments were performed on a Horiba DLS particle-size analyzer LB550. The data were collected at 25 °C by monitoring the scattered light intensity at a 90° detection angle. Each light scattering measurement was performed at least three times.
2.1. Materials 2.3. Cell culture and treatment C60 was purchased from Wuhan University (Hubei, China). RMPI 1640 medium, DMEM medium and fetal calf serum were provided from Gibco BRL (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl (MTT) was provided from Sigma (St. Louis, MO,
HeLa cells were cultured in an atmosphere with 5% CO2 and at 37 °C provided by a NAPCO CO2 incubator in RMPI 1640 medium containing 10% heat-inactivated fetal calf serum. All cells were
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cultured in poly-D-lysine coated culture dishes. The medium was changed every other day and cells were plated at an appropriate density according to each experimental scale. Then, the culture medium was replaced by the fresh medium containing 2% fetal calf serum and WSFD. For illumination of HeLa cells, we used a white light source (Lumacare, Newport Beach, CA) fitted with a light guide containing a bandpass filter (400–700 nm) adjusted to give a uniform spot of 4 cm in diameter with an irradiance of 54 mW/ cm2 as measured with a power meter. After 12 h incubation at 37 °C with WSFD, the cells were exposed to visible light at 25 °C. To avoid heat damage to the cells, the heat produced by the lamp was removed by a piece of heat-shield glass. Following irradiation, the plates were incubated for 24 h at 37 °C, 5% CO2. Each experiment was compared with a culture control without WSFD. The same procedure without irradiation was carried out to determine dark toxicity. To determine the tumor specific selectivity of WSFD, Neuro-2a (N2a) cells were cultured in the present study. N2a cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The medium was changed every other day and cells were plated at an appropriate density according to experimental scale. 2.4. Measurement of intracellular contents of WSFD The intracellular contents of WSFD were determined by fluorescence spectrometer (kexc = 380 nm, kem = 458 nm). The uptake of WSFD into HeLa cells or N2a cells was evaluated using a concentration of 5 lg/mL at different incubation times. In order to investigate the intracellular WSFD contents, cells of a certain number 106 cells/mL in PBS received freeze-thawing twice and were homogenized in the ice. The cell homogenate was centrifuged with 12,000 rpm for 15 min at 4 °C, and the supernatant was stored in the ice, then it was filtrated through a 0.2-mm membrane. The fluorescence intensity of the filtrate was determined by fluorescence spectrometer (FP-6500, Jasco, Japan).
WSFD, the cells were exposed to 32.4 J/cm2 visible light. After treatment, cells (1 106 cells per 3 mL in 6-well plates) were rinsed with D-Hanks solution and 10 lM DCF-DA was loaded. After 20 min incubation at 37 °C, the cells were harvested after being washed with PBS three times. The intracellular ROS accumulation was measured by using a Becton-Dickinson fluorescence-activated cell analyzer while data analysis was performed with Modifit LT 2.0 (Becton-Dickinson, San Jose, CA, USA). About 1 104 cells were counted for each analysis. The fluorescence intensity was quantified with CELLQuest software (Becton-Dickinson, Mountain View, CA, USA). 2.7. MTT reduction assay HeLa cells were plated at density of 1 104 cells per 100 lL in 96-well plates and the cell viability was determined by the conventional MTT reduction assay. After treatment, cells were treated with the 10 lL MTT solution (final concentration, 0.5 mg/mL) for 4 h. The dark blue formazan crystals formed in intact cells were solubilized with the lysis buffer (20% sodium dodecylsulfate in 50% aqueous N,N-dimethylformamide) and absorbance at 570/ 630 nm was measured with a microplate reader (Molecular Devices, Sunnylvale, CA, USA). 2.8. Determination of apoptosis Apoptotic cell death was analyzed by double staining with annexin V-FITC and propidium iodide (PI), in which annexin V bound to early apoptotic cells with exposed phosphatidylserine, while PI labeled the late apoptotic/necrotic cells with membrane damage. After 12 h incubation at 37 °C with WSFD, the cells were exposed to 32.4 J/cm2 visible light. After treatment, the apoptosis of cells was evaluated using an Annexin-V FITC apoptosis detection kit. Cells were harvested, washed and incubated at 4 °C for 30 min in the dark with annexin V-FITC and PI, then were analyzed on a FACS Vantage SE flow cytometer (Becton Dickinson, San Jose, CA, USA).
2.5. ROS detection in aqueous solutions 2.9. Determination of mitochondrial membrane potential The production of ROS was determined by measuring the intensity of green fluorescence emitted by redox-sensitive dyes dihydrorhodamine 123 (DHR; Sigma). After 32.4 J/cm2 visible light irradiation, the ROS generation in WSFD water mixture with DHR (5 mM) was assessed using fluorescence microplate reader (Chameleon; Hidex, Finland) equipped with a 488 nm excitation filter and a 535 nm emission filter. The results of ROS production by WSFD in water were presented as fold increase in DHR fluorescence in comparison with the control (water alone). The electron paramagnetic resonance (EPR) spectroscopy was used to monitor the generation of singlet oxygen in aqueous solutions. The EPR experiments were performed at room temperature on a Varian Eline spectrometer operating at a nominal frequency of 9.5 GHz. The aqueous solution containing 5 lg/mL of WSFD was supplemented with tetramethylpiperidine at 1 mM. Solutions were then exposed to illumination with visible light (32.4 J/cm2). Immediately after exposure to visible light, the sample-holding quartz tubes were transferred into the EPR cavity of the EPR spectrometer (Bruker BioSpin GmbH). Quantification of the signals was carried out by calculating the mean value of EPR signal amplitudes, and the data are expressed in arbitrary units. 2.6. Measurement of intracellular ROS accumulation The fluorescent probe DCF-DA was used to monitor the intracellular accumulation of ROS. After 12 h incubation at 37 °C with
HeLa cells were treated with WSFD for 12 h prior to visible light irradiation (32.4 J/cm2). After treatment, mitochondrial membrane potential (Dw) was assessed using DePsipher (R&D Systems), a lipophilic cation that had the property of aggregating upon membrane polarization forming an orange-red fluorescent compound. If the potential was disturbed, the dye cannot access the transmembrane space and remains or reverts to its green monomeric form. The cells were stained with DePsipher as described by the manufacturer, and the green monomer and the red aggregates were detected by flow cytometry. The results were presented as a red/green fluorescence ratio (FL2/FL1, arbitrarily set to 100% in untreated control samples), the increase and decrease of which reflect mitochondrial hyperpolarization and depolarization, respectively. 2.10. Measurement of caspase-3 activity HeLa cells were treated with WSFD for 12 h prior to visible light irradiation (32.4 J/cm2), and the caspase-3 activity was measured according to the manufacturer’s protocol. In brief, cells were lysed for 10 min in an ice bath, and centrifuged at 15,000g for 10 min at 4 °C, then the supernatant were incubated with acetyl-Asp-GluVal-Asp-aldehyde-AFC at 37 °C for 1 h. Fluorescence intensity was measured using a Jasco FP-6500 fluorescence spectrophotometer (kex 400 nm and kem 505 nm). The value for each treatment group was converted to the percentage of control.
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2.11. Measurement of MDA content and antioxidant enzyme activities For assay of MDA and antioxidant enzyme, the cultures were washed two times with phosphate-buffered saline (PBS), then scraped from the plates into ice cold PBS (0.1 M, containing 0.05 mM ethylene diamine tetraacetic acid) and homogenized. The homogenate was centrifuged at 4 °C at 10,000g for 30 min. The resulting supernatant was stored at 70 °C until the following analyses. Protein concentration was determined by the Bradford method, using bovine serum albumin as a reference standard. HeLa cells were treated with WSFD for 12 h prior to visible light irradiation (32.4 J/cm2). After 24 h incubation, the content of MDA was determined using the thiobarbituric acid method. Antioxidant enzyme activities were assayed after cells were incubated with WSFD for 12 h then exposed to 32.4 J/cm2 visible light. The processes of measurement were according to the instructions for the reagent kits. 2.12. Statistical analysis Values are reported as means ± S.D. Statistical comparisons were made by one-way ANOVA to detect significant difference using SPSS 13.0 for windows. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Characterization of WSFD In this work, amino group of amino acid and folic acid reacted with C60 to form the products. Analytic data of the synthesized and purified compounds were shown below. PFD: IR(KBr) m: 3421, 1709, 1604, 1496, 1068, 698, 579, 530 (C60 core), cm 1; 1H NMR(D2O) dppm: 7.21 (m), 4.02 (s), 3.43 (m), 2.84, 1.08 (m, C60–H); 13C NMR(D2O) dppm: 173.3, 138.7 (m, C60), 128.4, 57.5, 38.2; ESI-MS m/z (%): 1545 (M+, 34.6), 720 (100). Anal. calcd for C105H55N5O10: C 81.55, H 3.56, N 10.36; found C 80.87, H 3.87, N 10.56. The results indicated that there were five phenylalanine moieties per C60 molecule. FFD: IR(KBr) m: 3501, 3382, 2976, 1691, 1601, 1563, 1524, 1450, 1387, 1124, 1189, 570, 527 (C60 core), cm 1; 1H NMR(D2O) dppm: 9.0 (s), 7.7 (d), 6.8 (d), 4.3 (s), 3.6 (d), 2.3 (d), 2.1 (s), 1.9 (s), 1.2 (m, C60–H); 13C NMR(D2O) dppm: 174.9, 173.7, 161.1, 156.5, 150.7, 139.8–150.8 (m, C60), 118.6, 106.0, 55.6, 41.6, 31.2, 26.0; ESI-MS m/z (%): 1161 (M+, 38), 720 (100). Anal. calcd for C79H19N7O6: C 81.65, H 1.64, N 8.44; found C 79.76, H 1.87, N 8.93. The reaction on fullerene gave mono-adduct demonstrated by mass spectroscopy and elemental analysis. AFD: IR(KBr) m: 3430, 3187, 1638, 1574, 1401, 1190, 527 (C60 core), cm 1; 1H NMR(D2O) dppm: 3.58 (m), 2.84 (m), 2.02 (s),
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1.11 (m, C60–H); 13C NMR(D2O) dppm: 170.3, 137.0–155.0 (m, C60), 153.2, 77.0, 51.2, 39.8, 25.7, 23.6. ESI-MS m/z (%): 2112 (M+, 45.1), 720 (100). Anal. calcd for C108H112N32O16: C 61.36, H 5.30, N 21.21; found C 60.08, H 5.38, N 21.78. The results indicated that there were eight arginine moieties per C60 molecule of AFD. The intensity size distribution was measured by DLS particle-size analyzer (Fig. 2). The average hydrodynamic diameter of 1 mg/mL PFD, FFD and AFD was 5.5, 120.1 and 779.2 nm, respectively. 3.2. Assays of intracellular uptake of WSFD The fluorescence quantum yield of C60 was very low. Therefore, it was difficult to observe strong fluorescence of isolated C60 at normal experimental conditions. In aqueous solution of WSFD, interaction between adducts and C60 distorted the icosahedral symmetry of C60. The relaxation of selection rules due to the reduced symmetry of C60 molecule induced the observed strong fluorescence peaks [32]. The uptake of WSFD into HeLa cells was evaluated using a concentration of 5 lg/mL at different incubation times. In each case, the WSFD uptake was determined by fluorescence analysis. The results were summarized in Fig. 3A. WSFD was rapidly incorporated in the HeLa cells in the initial time (<4 h) and the uptake tended to a saturation value between 4 and 24 h. This value was estimated as 2.24, 2.98 and 0.85 lg per 106 cells for PFD, FFD and AFD, respectively. The result in our experiment demonstrated that WSFD could penetrate through the cell membrane to retain molecular stability, which further exhibited photodynamic activities in cytoplasm. The function groups with tumor targeting increased the uptake of WSFD in HeLa cells. In the same experiment condition, The N2a uptake of WSFD was estimated as 1.52, 1.37 and 0.41 lg per 106 cells for PFD, FFD and AFD, respectively. The uptake of WSFD in N2a cells was much less than HeLa cells (Fig. 3B). 3.3. ROS production by WSFD As it has previously been demonstrated that the cytotoxicity of WSFD was ROS mediated, we compared the ability of different WSFD to produce ROS. The data of DHR measurement of dosedependent ROS production in a cell free system (Fig. 4A) revealed a positive correlation between the cytotoxic capacity of the WSFD and their capacity to produce reactive oxygen. The EPR spectra of different WSFD showed that very strong TEMPOL signal was generated by PFD and FFD, while marginal signal was observed with AFD (Fig. 4B and C). The EPR data for the first time demonstrated a singlet oxygen production in water solutions of WSFD. However, it should be noted that the reactivity of non-specific ROS reporter dyes DHR indicated that other active oxygen species such as superoxide anion (O2 ), might also be generated in water solutions of WSFD. Accumulation of intracellular ROS was detected by using DCFDA. The cytotoxicity of WSFD towards HeLa cells was associated with an increase in the intracellular ROS production (Fig. 4D). When 5 lg/mL WSFD was irradiated by visible light, intracellular ROS accumulation was increased from 103.2 to 456.8, 512.9 and 198.6, respectively, which was determined by changes in fluorescence intensity. The same procedure without irradiation was carried out to determine the ROS generation in dark. The results of the intracellular ROS measurement, however, showed that FFD possessed the highest ROS-generating power. 3.4. WSFD-induced cytotoxicity and apoptosis in HeLa cells
Fig. 2. Size distribution functions of the colloidal dispersions formed by WSFD in water. The concentration of WSFD in the water was 1 mg/mL.
After 12 h incubation with WSFD followed by light irradiation, the phototoxicity of WSFD was tested by the MTT assay (Fig. 5A). The results showed that the decrease in cell viability was dependent on the irradiation time. The longer irradiation time, the higher
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Fig. 3. (A) Uptake of WSFD into HeLa cells as a function of incubation time. (B) Uptake of WSFD into N2a cells as a function of incubation time. The uptake of WSFD into cells was evaluated using a concentration of 5 lg/mL incubated for different incubation time. Values represent mean ± S.D. (n = 3).
Fig. 4. ROS production by WSFD. (A) Concentration-dependent ROS production by WSFD in water, measured as increase in DHR fluorescence. (B) The representative EPR spectra of 1O2 generation by WSFD. (C) EPR measurement of 1O2 generation by WSFD in comparison with control sample containing H2O. (D) Intracellular ROS generation by WSFD. The results from representative of at least three experiments are presented. Values represent mean ± S.D. (n = 3). ⁄p < 0.05 compared to the control group.
photodynamic inactivation of cell viability was found. The viability of cells pretreated with PFD, FFD and AFD at 5 lg/mL followed by 32.4 J/cm2 light irradiation was decreased to 44.2%, 27.4% and 76.4% of control value, respectively. The phototoxicity of WSFD presented the same trend as the light-induced ROS production, which confirmed the photodynamic effects of WSFD were resulted from their ability to generate ROS when photoexcited. Cell death was not observed when non-treated cell cultures were irradiated with visible light under the above described condition. Therefore, cell death of the cultures treated with the WSFD and then irradiated was caused by both visible light and the PS effect.
The next set of experiments was aimed at gaining some additional insight into the mechanisms of the phototoxic action of WSFD. We performed double staining of HeLa cells with annexin V-FITC and PI to detect phosphatidylserine exposure (annexin V) and cell membrane damage (PI). In control groups, 2.23% cells excluded PI and were positive for annexin V-FITC binding, which represented apoptotic cells. After 12 h incubation with 5 lg/mL WSFD followed by light irradiation (32.4 J/cm2), the percentage of apoptosis increased to 39.4%, 52.8% and 10.7%, respectively (Fig. 5B). In addition to apoptotic cells, the percentage of necrotic cells was 14.5%, 16.9% and 8.7% for PFD, FFD and AFD, respectively.
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Fig. 5. WSFD-induced cytotoxicity and apoptosis in HeLa cells. (A) HeLa cells were incubated with 5 lg/mL WSFD for 12 h prior to visible light irradiation (54 mW/cm2, different irradiated time). Cell viability was measured by the conventional MTT reduction assay. Data were presented as mean ± S.D. (n = 3). (B) HeLa cells were incubated with 5 lg/mL WSFD for 12 h prior to visible light irradiation (32.4 J/cm2). WSFD-induced apoptosis was determined by flow cytometry. Cells incubated with 5 lg/mL WSFD without irradiation served as dark group. Data were presented as mean ± S.D. (n = 3). ⁄p < 0.05 compared to the control group.
The results indicated that apoptotic cell death was predominant in all experiments. This was an advantageous result since apoptosis was an ordered process during an organism’s life cycle and took place without the drastic effect of inflammation as observed for necrosis. Our results also showed that 5 lg/mL WSFD didn’t induce dark cytotoxicity. 3.5. WSFD-induced apoptotic events The oxidation of membrane lipids, one of the primary events in oxidative cellular damage, can be assessed by measurement of MDA, a breakdown product of lipid peroxides. The cytotoxicity caused by ROS normally accompanied by the increase of lipid peroxides. Treatment of HeLa cells with WSFD followed by light irradiation caused significant increase in the intracellular MDA level (Table 1). PFD, FFD and AFD (5 lg/mL) increased the MDA level to 207.1%, 262.5% and 155.4% of control, respectively. The depolarization of mitochondrial membrane, an initial event in apoptosis induction, was readily detected after the light irradiation in the presence of WSFD, as demonstrated by a decrease in the red/green (FL2/FL1) fluorescence ratio of mitochondrial-binding dye DePsipher (Table 1). Treatment with WSFD followed by light irradiation significantly decreased the value of Dw. PFD, FFD and AFD (5 lg/mL) decreased the Dw to 44.6%, 34.8% and 81.4% of control, respectively. To further confirm the observation that WSFD induced apoptosis, caspase-3 activity was tested. Treatment with WSFD followed by irradiation significantly increased caspase-3 activity (Table 1), an enzyme family involved in apoptosis execution. Our results also showed that addition of WSFD without irradiation didn’t affect MDA level, Dw and caspase-3 activity (Table 2).
Table 1 Photodynamic effects of WSFD on MDA, mitochondrial membrane potential and caspase-3 activity in HeLa cells. Treatment
MDA (nmol/mg protein)
Dw (% of control)
Caspase-3 activity (% of control)
Control PFD FFD AFD
0.56 ± 0.08 1.16 ± 0.17* 1.47 ± 0.13 0.87 ± 0.09*
100.0 ± 5.1 44.6 ± 5.6* 34.8 ± 6.4* 81.4 ± 3.9*
100.0 ± 4.8 216.7 ± 10.8* 264.3 ± 8.9* 156.3 ± 11.6*
HeLa cells were incubated with WSFD (5 lg/mL) for 12 h prior to visible light irradiation (32.4 J/cm2). Data are presented as mean ± S.D. (n = 3). * p < 0.05 compared to the control group.
Table 2 Dark cytotoxicities of WSFD on MDA, mitochondrial membrane potential, caspase-3 activity and antioxidant enzyme activities in HeLa cells. Chemicals
PFD
FFD
AFD
MDA (nmol/mg protein) Dw (% of control) Caspase-3 (% of control) SOD (U/mg protein) CAT (U/mg protein) GSH-Px (U/mg protein)
0.54 ± 0.11 98.6 ± 3.2 104.6 ± 3.8 55.8 ± 3.7 26.7 ± 3.3 23.6 ± 3.8
0.52 ± 0.07 101.3 ± 4.9 105.3 ± 6.7 53.9 ± 4.3 26.3 ± 4.3 27.9 ± 3.9
0.55 ± 0.07 96.8 ± 5.3 102.9 ± 2.8 55.6 ± 5.9 25.4 ± 3.4 24.7 ± 2.7
HeLa cells were incubated with WSFD (5 lg/mL) for 36 h without visible light irradiation. Data are presented as mean ± S.D. (n = 3).
Table 3 Photodynamic effects of WSFD on antioxidant enzyme activities in HeLa cells. Treatment
SOD (U/mg protein)
CAT (U/mg protein)
GSH-Px (U/mg protein)
Control PFD FFD AFD
52.5 ± 4.1 16.3 ± 2.1* 9.8 ± 1.2* 30.8 ± 2.5*
23.6 ± 3.1 10.4 ± 0.7* 5.8 ± 0.7* 15.8 ± 2.1*
25.7 ± 2.6 8.9 ± 1.1* 4.8 ± 0.6* 16.1 ± 1.7*
HeLa cells were incubated with WSFD (5 lg/mL) for 12 h prior to visible light irradiation (32.4 J/cm2). Data are presented as mean ± S.D. (n = 3). * p < 0.05 compared to the control group.
3.6. WSFD caused loss of antioxidant enzyme activities in HeLa cells HeLa cells treated with WSFD followed by irradiation caused the decrease in the activity of SOD, CAT and GSH-Px (Table 3). Take FFD for example, cells treated with FFD and then irradiated caused the decrease in the activity of SOD, CAT and GSH-Px by 81.3%, 75.4% and 81.3%, respectively. Again, treatment with WSFD without irradiation didn’t alter the activities of SOD, CAT and GSH-Px in cultured HeLa cells (Table 2). 4. Discussion The potent ability of fullerene and its derivatives to photosensitize transition of molecular oxygen to highly reactive ROS makes them promising candidates for the photodynamic killing of cancer cells. The main advantage of this therapeutic approach is selectivity, achieved by tumor-specific activation of photosensitizing agent
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by highly focused light beam delivered to tumor region at the surface of the body or to internal tumors using optical fibers [33]. The effectiveness of various PS proposed for anticancer PDT can be judged on several criteria. The PS should be able to kill multiple classes of cancer cells at relatively low concentrations. PS should be reasonably non-toxic in the dark and should demonstrate selectivity for cancer cells over normal cells. The PS should actually be taken up inside the cell, and that the generation of ROS outside the cell will not be sufficient to produce efficient cell death. In present study, we have synthesized three WSFD, which have the potential to selectively kill cancer cells without dark toxicity. It was found that 5 lg/mL WSFD caused significant cell death after 10 min visible light irradiation. It should be noted that the photodynamic effect was apparently not restricted to HeLa cell line used in the present study, since it was also observed in other tumor cell lines, such as human laryngeal squamous cell carcinoma strain Hep-2 (Hu et al. unpublished observation). The clustering of WSFD in the aqueous phase, in which it was suggested to form micellar aggregate with the hydrophobic C60 core in the center and the hydrophilic groups were sticking into the water phase. The photodynamic effects of WSFD were associated primarily with conjugated molecular structure of C60. However, the morphology of the aggregate inhibited the photon energy absorbing and ROS generation of WSFD. The aggregation morphology of WSFD was mainly associated with the hydrophobic interaction and hydrogen bond. Both the chemical structure of adducts and addend numbers influenced the aggregate size of WSFD. In this work, the aggregate sizes of FFD and AFD were larger than PFD (AFD > FFD > PFD). It could be explained by considering the effect of hydrogen bond. FFD and AFD contained multiple amino and carboxylic groups within the same molecular framework, which self-assembled to form larger aggregates that were organized through bidentate hydrogen bonding interaction. However, the hydrophilic heads of PFD contained only one imino and one carboxylic group, which resulted in hydrogen bond hard to be formed between molecules. The hypothetic mechanism of the WSFD
self-aggregates was shown in Fig. 6. The data presented here on the PFD also showed that several hydrophilic addends were sufficient to prevent the strong hydrophobic three dimensional interactions among the fullerene moieties and the resulting tendency to form aggregates. Investigators demonstrated that both physical properties and chemical properties influenced the biological and biomedical activities of functionalized fullerenes [34]. The first factor contributing to radical-generation activity (i.e., 1O2 and O2 ) was the aggregate structure of WSFD. In present study, it was found that the ROS generation of WSFD in water (PFD > FFD > AFD) tended to decrease with increasing of aggregate sizes. In addition to chemical and physical properties of C60, other factors [35], such as cellular uptake, might profoundly influence the phototoxicity of C60-based agents. The results of intracellular ROS generation indicated by a disagreement of the ROS producing order (FFD > PFD > AFD) with the results got from ROS generation in cell free system (PFD > FFD > AFD). Since FFD possessed the highest uptake in HeLa cells, it could generate most ROS in cytoplasm. An important feature of various C60 preparations that facilitates their biological reactivity was the ability to penetrate cell membrane and gain access to cell cytoplasm, organelles and nucleus, as predicted by theoretical studies and confirmed in various experimental settings [36–38]. In present study, the WSFD uptake was determined by fluorescence analysis. The results indicated that the cellular uptake of WSFD was associated with functional groups. The proliferation rate of tumor cells is much faster than normal cells. Avid uptake of amino acids is a normal feature of rapidly proliferating cells. The membrane-associated folic acid receptor is a tumor marker that is overexpressed on a variety of neoplastic tissues, including breast, cervical, ovarian, colorectal, renal and nasopharyngeal tumors, but highly restricted in most normal tissues. Thus, both amino acids and folic acid can be used as targeting carrier for anticancer drugs. When amino acids and folic acid were covalently linked to C60 by amino moiety, its affinity for its cell surface receptor remained essentially unaltered. Further, following binding to
Fig. 6. The hypothetic mechanism of the WSFD self-aggregates in water.
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Fig. 7. The hypothetical mechanism of the photodynamic activities of WSFD. To overcome the poor solubility, biocompatibility and selectivity of C60, C60 was modified with Lphenylalanine, folic acid and L-arginine. As designed, the WSFD had good selectivity to tumor cells without dark cytotoxicity. After cell uptake and visible light irradiation, WSFD generated the 1O2 and other ROS (O2 ) in vitro. In parallel, following exposure of cells to WSFD and irradiation, a marked decrease in mitochondrial membrane potential, activities of SOD, CAT and GSH-Px were observed. Moreover, WSFD caused significant elevation in caspase-3 activity, and induced apoptotic death.
the cell surface receptor, the WSFD were internalized by cell. The sequence of the uptake of WSFD was FFD > PFD > AFD, which indicated folic acid had the best targeting among the three functional groups. Furthermore, the cellular uptake of WSFD in HeLa cells was much more than N2a cells, which indicated that WSFD had excellent selectivity to tumor cells. There two different mechanisms can explain the formation of cytotoxic species. Type I mechanism proceeds through formation of free radicals or O2 resulting from electron or hydrogen transfer. Type II mechanism is a process of the resonant energy transfer occurring during collisions between molecules of oxygen and sensitizer. In this process, the energy captured by a sensitizer is transferred to dioxygen, thus giving rise to generation of 1O2. The singlet excited state of C60 (1C60 ), initially formed upon light excitation, undergoes intersystem crossing to triplet state (3C60 ) that can be efficiently quenched by molecular oxygen to generate large amounts of 1O2. In the present study, the EPR data demonstrate the 1O2 production in water solutions of WSFD. This result indicates that the WSFD generates ROS by Type II mechanism. However, photoexcited C60 also efficiently mediates electron transfer from common electron donors (e.g., nicotinamide adenine dinucleotide) to electron acceptors such as oxygen to produce O2 . In the present study, the DHR assay data indicate that other active oxygen species such as O2 , might also be generated in water solutions of WSFD. This result indicates that the WSFD may also generate ROS by Type I mechanism. Thus, the WSFD generates ROS both by Type I and Type II mechanism in the photosensitization process. These unique photochemical properties have expanded WSFD applications into PDT. The oxidation of membrane lipids can be assessed by measurement of MDA. On the other hand, cells are often equipped with several antioxidant mechanisms (SOD, CAT and GSH-Px) that serve as a detoxifying system to prevent damage
caused by oxidative stress. The present study proved that cells treated with WSFD followed by light irradiation caused a marked decrease in cell survival, elevation of oxidative stress characterized by MTT and MDA production, and reduction in SOD, CAT and GSHPx activities. In addition to producing consequent lipid peroxidation and depolarization of mitochondrial membrane, WSFD exposure caused rising caspase-3 activity. Thus, the photodynamic effects of WSFD apparently involved induction of the ‘‘programmed’’ cell death, known as apoptosis [16]. This was consistent with the preferential mitochondrial localization of WSFD [36], having in mind that ROS induced mitochondrial dysfunction was a key initial step in the ‘‘mitochondrial’’ pathway of apoptosis [39]. The hypothetical mechanism was shown in Fig. 7. This study demonstrated that the photodynamic effects of fullerenes were linked to ROS generation under irradiation, which should be valuable for developing highly effective fullerene derivatives for use in biomedicine.
5. Conclusions We have synthesized three WSFD, which had tumor target photokilling activities without dark toxicity on HeLa cells. Both chemical properties and physical properties influenced the ROSgeneration abilities, cellular uptake and photodynamic activities of WSFD. The WSFD exerted photodynamic effects by targeted uptake and 1O2/O2 generation under irradiation with the following relative potencies: FFD > PFD > AFD. Pretreatment of the cells with WSFD followed by visible light irradiation induced cell apoptosis noticeably, such as oxidation of membrane lipids, depolarization of mitochondrial membrane, SOD, CAT and GSH-Px activities reduction and subsequent caspase-mediated apoptotic cell death.
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Furthermore, this work also provides a new way to synthesize novel fullerene derivatives with anticancer properties. Conflict of interest statement None declared. Acknowledgments The project was supported by National Natural Science Foundation of China (No. 51103031), Special Foundation of China Postdoctoral Science (No. 201003436), the Fundamental Research Funds for the Central Universities (No. HIT. NSRIF. 2012034) Science and Technology Projects Fund for Innovative Talent of Harbin (No. 2009RFQXG044). References [1] J.P. Celli, B.Q. Spring, I. Rizvi, C.L. Evans, K.S. Samkoe, S. Verma, B.W. Pogue, T. Hasan, Imaging and photodynamic therapy: mechanisms, monitoring, and optimization, Chem. Rev. 11 (2010) 2795–2838. [2] C.L. Peng, P.S. Lai, F.H. Lin, S.Y.H. Wu, M.J. Shieh, Dual chemotherapy and photodynamic therapy in an HT-29 human colon cancer xenograft model using SN-38-loaded chlorin-core star block copolymer micelles, Biomaterials 30 (2009) 3614–3625. [3] J. Schwiertz, A. Wiehe, S. Gräfe, B. Gitter, M. Epple, Calcium phosphate nanoparticles as efficient carriers for photodynamic therapy against cells and bacteria, Biomaterials 30 (2009) 3324–3331. [4] B. Bae, K. Na, Self-quenching polysaccharide-based nanogels of pullulan/folatephotosensitizer conjugates for photodynamic therapy, Biomaterials 31 (2010) 6325–6335. [5] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, M.J. Thun, Cancer statistics, CA-Cancer J. Clin. 59 (2009) 225–249. [6] Z. Markovic, V. Trajkovic, Biomedical potential of the reactive oxygen species generation and quenching by fullerenes C60, Biomaterials 29 (2008) 3561– 3573. [7] N.S. Zogovic, N.S. Nikolic, S.D. Vranjes-Djuric, L.M. Harhaji, L.M. Vucicevic, K.D. Janjetovic, M.S. Misirkic, B.M. Todorovic-Markovic, Z.M. Markovic, S.K. Milonjic, V.S. Trajkovic, Opposite effects of nanocrystalline fullerene (C60) on tumour cell growth in vitro and in vivo and a possible role of immunosuppression in the cancer-promoting activity of C60, Biomaterials 30 (2009) 6940–6946. [8] P. Mroz, G.P. Tegos, H. Gali, T. Wharton, T. Sarna, M.R. Hamblin, Photodynamic therapy with fullerenes, Photochem. Photobiol. Sci. 6 (2007) 1139–1149. [9] Z. Markovic, B. Todorovic-Markovic, D. Kleut, N. Nikolic, S. Vranjes-Djuric, M. Misirkic, L. Vucicevic, K. Janjetovic, A. Isakovic, L. Harhaji, B. Babic-Stojic, M. Dramicanin, V. Trajkovic, The mechanism of cell-damaging reactive oxygen generation by colloidal fullerenes, Biomaterials 28 (2007) 5437–5448. [10] N. Singh, B. Manshian, G.J.S. Jenkins, S.M. Griffiths, P.M. Williams, T.G.G. Maffeis, C.J. Wright, S.H. Doak, NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials, Biomaterials 30 (2009) 3891–3914. [11] Y. Liu, Y.L. Zhao, Y. Chen, P. Liang, L. Li, A water-soluble b-cyclodextrin derivative possessing a fullerene tether as an efficient photodriven DNAcleavage reagent, Tetrahedron Lett. 46 (2005) 2507–2511. [12] I.M. Belousova, O.B. Danilov, T.D. Murav’eva, I.M. Kiselyakov, V.V. Ryl’kov, T.K. Kris’ko, T.D. Murav’eva, Solid-phase photosensitizers based on fullerene C60 for photodynamic inactivation of viruses in biological liquids, J. Opt. Technol. 76 (2009) 243–250. [13] V.V. Zarubaev, I.M. Belousova, O.I. Kiselev, L.B. Piotrovsky, P.M. Anfimov, T.C. Krisko, T.D. Muraviovab, V.V. Rylkovb, A.M. Starodubzevb, A.C. Sirotkina, Photodynamic inactivation of influenza virus with fullerene C60 suspension in allantoic fluid, Photodiagn. Photodyn. Ther. 4 (2007) 31–35. [14] J.P. Kamat, T.P. Devasagayam, K.I. Priyadarsini, H. Mohan, Reactive oxygen species mediated membrane damage induced by fullerene derivatives and its possible biological implications, Toxicology 155 (2000) 55–61. [15] F. Rancan, S. Rosan, F. Boehm, A. Cantrell, M. Brellreich, H. Schoenberger, A. Hirsch, F. Moussa, Cytotoxicity and photocytotoxicity of a dendritic C(60) mono-adduct and a malonic acid C(60) trisadduct on Jurkat cells, J. Photochem. Photobiol. B 67 (2002) 157–162.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Photodynamic anticancer activities of water-soluble C60 derivatives and their biological consequences in a HeLa cell line Zhen Hu a,⇑, Chunhua Zhang a, Yudong Huang a,⇑, Shaofan Sun a, Wenchao Guan b, Yuhuan Yao a a b
School of Chemical Engineering and Technology, Harbin Institute of Technology, Harbin 150001, China College of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e
i n f o
Article history: Received 29 August 2011 Received in revised form 10 October 2011 Accepted 7 November 2011 Available online 15 November 2011 Keywords: Fullerene Photosensitization Free radical Antitumor Apoptosis
a b s t r a c t Photodynamic therapy is an emerging, externally activatable, treatment modality for various diseases, especially for cancer therapy. The photodynamic activities of tumor targeting water-soluble C60 derivatives (WSFD) were evaluated on HeLa cells. To overcome the poor solubility, biocompatibility and selectivity of C60, we modified C60 with L-phenylalanine, folic acid and L-arginine. Consistent with their photodynamic abilities, WSFD generated the reactive oxygen species after irradiation both in water and in vitro. No dark cytotoxicity was observed using 5 lg/mL WSFD during long incubation time. Furthermore, the uptake of WSFD into HeLa cells was much more than normal cells, which indicated the WSFD had selectivity to tumor cells. Investigation of the possible photodynamic activities of WSFD demonstrated that they expressed photokilling activities by raising the level of 1O2/O2 under visible light irradiation. In parallel, following exposure of cells to WSFD and irradiation, a marked decrease in mitochondrial membrane potential, cell viability, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased malondialdehyde (MDA) production were observed. Moreover, WSFD caused significant elevation in caspase-3 activity, and induced apoptotic death. Experiments demonstrated that both chemical properties, such as the chemical structure of adduct and addend numbers, and physical properties, such as degree of aggregation, influenced the ROS-generation abilities, cellular uptake and photodynamic activities of WSFD. The results suggest that WSFD have the potential application in cancer cell inactivation by photodynamic therapy. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Photodynamic therapy (PDT) is an emerging, externally activatable, treatment modality for various diseases. The use of PDT as a cancer therapy is particularly attractive because of its fundamental specificity and selectivity [1–4]. This is due to the fact that the photosensitizer (PS) concentrates specifically within the malignant tissue so when the light is directly focused on the lesion, it causes reactive oxygen species (ROS) to be generated resulting in cellular destruction at the region of interest. For this reason, in recent years, PDT has become the subject of intense investigation as a possible treatment modality for various forms of cancer. Similar to chemotherapy, PDT still requires agents which exhibit selectivity for the target cells. Similar to radiotherapy, the mode of action with PDT involves the use of electromagnetic radiation in order to generate radical species in situ. However, PDT is a much milder approach for cancer treatment than either [5]. The reason for this lies ⇑ Corresponding authors. Tel.: +86 451 86413711; fax: +86 451 86402403 (Z. Hu). E-mail addresses: (Y. Huang).
[email protected] (Z. Hu),
[email protected]
0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.11.003
in the combination of the mode of action of the PS employed and its activation in situ by relatively long wavelength, visible light. Because of their unique physicochemical properties, fullerenes (C60) are potential candidates for many biomedical applications, including PDT [6,7]. The anticancer properties of various fullerene preparations usually result from their ability to generate cytotoxic ROS when photoexcited, which is the main principle of PDT [8,9]. Various fullerenes, including pristine C60 as well as functionalized derivatives, have been previously used to carry out PDT reactions leading to cleavage of DNA strands [10,11], photoinactivation of viruses [12,13], production of oxidative damage to lipids in microsomal membranes [14], PDT-induced killing of mammalian cells in tissue culture [15,16], and even a report of regressions after PDT in a mouse tumor model [17]. Despite their potential, most C60 derivatives used in PDT have some limitations. Mainly, they are hydrophobic or have limited water solubility which leads to the modification of their optical properties and the decrease of singlet oxygen production. Although recently a lot of work has focused on developing several new strategies to improve the solubility of C60 derivatives, including modification of the fullerenes with watersoluble substituents [18–20] and solubilization using polymers
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Fig. 1. The chemical structures of (A) PFD, (B) FFD and (C) AFD.
[21], lipid membranes [22], cyclodextrin [23], calixarenes [24], the lack of specific target and the dark cytotoxicity is still a principal challenge for C60 derivatives. In order to solve the problem described above, C60 was modified by L-phenylalanine, folic acid and L-arginine in this work. L-Phenylalanine and L-arginine are necessary amino acids for human body, which have been widely used in pharmaceutical and food additive industry. It has been reported that L-phenylalanine can be used as carrier for anticancer drugs, which delivers the drug molecules into the tumor area directly [25,26]. Its transfer effect is 3–5 times higher than other amino acids. Preliminary evidence surrounding the use of folic acid seems promising for decreasing the risk of breast, cervical, pancreatic and gastrointestinal cancer. Furthermore, the discovery of high-levels expression of folic acid receptor on many human cancer cells has rendered the folic acid as an attractive candidate for development of tumor specific therapeutics [27–29]. Thus, the modifiers we chose in this work possessed good biocompatibility and targeting. The opportunity of combining C60 with Lphenylalanine, folic acid and L-arginine appears as a desirable way to develop novel PS for PDT (Fig. 1). As designed, the PS will have good selectivity to tumor cells without dark cytotoxicity. In our previous study, we have reported that water-soluble C60 derivatives could play important role in protecting cells from apoptosis induced by hydrogen peroxide and nitric oxide [30,31]. On the other hand, illumination of C60 with visible or UV light fosters its transition to a long-lived triplet excited state and the subsequent energy transfer to molecular oxygen, yielding ROS. This dual property of C60 to either quench or generate cell-damaging ROS could be therefore exploited for its development as a cytoprotective or cytotoxic anticancer agent [6]. In this work, we have synthesized three water-soluble C60 derivatives (WSFD) by nucleophilic addition reaction. The compounds were characterized by Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), liquid chromatography–mass spectrometry (LC–MS), elemental analysis and dynamic light scattering (DLS). The photodynamic effects were evaluated on HeLa human cervix uteri tumor-derived cell line. The ROS production ability of WSFD was analyzed both in cell free condition and in vitro. After incubation with WSFD and visible light irradiation the mechanisms of cellular death induced by the derivatives were studied. 2. Experimental
USA). 2,7-Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probe Inc. (Eugene, OR, USA). The reagent kits for the measurement of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ApoAlert caspase fluorescent assay kit was purchased from BD Bioscience Clontech (Palo Alto, CA, USA). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from Biosea BCL (Beijing, China). L-Phenylalanine, folic acid and L-arginine were products of Sinoreagent Co. Ltd. (Shanghai, China). 2.2. The synthesis and characterization of WSFD WSFD was synthesized by nucleophilic addition reaction. L-Phenylalanine, folic acid or L-arginine (10 mmol) and sodium hydroxide (20 mmol) were dissolved in 3 mL water, and then 30 mL ethanol was added, the resulting solution was added to a C60 toluene solution (0.1 mmol, 60 mL) dropwise, then 5 drops of 10% tetrabutylammonium hydroxide was added under stirring. The solution was stirred at room temperature under nitrogen atmosphere. To make sure the reaction was completed, the solution was stirred for 48 h. The aqueous layer was separated from the organic layer, filtered, diluted with 3 mL water. Ethanol 40 mL was then added to cause the precipitation of the product, which was further reprecipitated with H2O/ EtOH for several times. Then the product was further purified by gel exclusion chromatography using a dextran (G-25, Pharmacia biotech) column with H2O. The product was eluted first, and then unreacted L-phenylalanine, folic acid or L-arginine was eluted. The resulting solution was dried under vacuum to give the product of L-phenylalanine C60 derivatives (PFD), folic acid C60 derivatives (FFD) or L-arginine C60 derivatives (AFD) (82.7%, 75.3% and 83.6% based on converted C60, respectively). The chemical structures of WSFD were characterized by FT-IR, 1 H NMR, 13C NMR, LC–MS and elemental analysis. The aggregation properties of WSFD were characterized by DLS. The preparation of WSFD vesicles were done by dissolving WSFD (100 mg) in H2O (100 mL) to form a solution of 1 mg/mL in concentration with ultrasonication for 2 h, followed by centrifugation and decantation. Light scattering experiments were performed on a Horiba DLS particle-size analyzer LB550. The data were collected at 25 °C by monitoring the scattered light intensity at a 90° detection angle. Each light scattering measurement was performed at least three times.
2.1. Materials 2.3. Cell culture and treatment C60 was purchased from Wuhan University (Hubei, China). RMPI 1640 medium, DMEM medium and fetal calf serum were provided from Gibco BRL (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl (MTT) was provided from Sigma (St. Louis, MO,
HeLa cells were cultured in an atmosphere with 5% CO2 and at 37 °C provided by a NAPCO CO2 incubator in RMPI 1640 medium containing 10% heat-inactivated fetal calf serum. All cells were
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cultured in poly-D-lysine coated culture dishes. The medium was changed every other day and cells were plated at an appropriate density according to each experimental scale. Then, the culture medium was replaced by the fresh medium containing 2% fetal calf serum and WSFD. For illumination of HeLa cells, we used a white light source (Lumacare, Newport Beach, CA) fitted with a light guide containing a bandpass filter (400–700 nm) adjusted to give a uniform spot of 4 cm in diameter with an irradiance of 54 mW/ cm2 as measured with a power meter. After 12 h incubation at 37 °C with WSFD, the cells were exposed to visible light at 25 °C. To avoid heat damage to the cells, the heat produced by the lamp was removed by a piece of heat-shield glass. Following irradiation, the plates were incubated for 24 h at 37 °C, 5% CO2. Each experiment was compared with a culture control without WSFD. The same procedure without irradiation was carried out to determine dark toxicity. To determine the tumor specific selectivity of WSFD, Neuro-2a (N2a) cells were cultured in the present study. N2a cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The medium was changed every other day and cells were plated at an appropriate density according to experimental scale. 2.4. Measurement of intracellular contents of WSFD The intracellular contents of WSFD were determined by fluorescence spectrometer (kexc = 380 nm, kem = 458 nm). The uptake of WSFD into HeLa cells or N2a cells was evaluated using a concentration of 5 lg/mL at different incubation times. In order to investigate the intracellular WSFD contents, cells of a certain number 106 cells/mL in PBS received freeze-thawing twice and were homogenized in the ice. The cell homogenate was centrifuged with 12,000 rpm for 15 min at 4 °C, and the supernatant was stored in the ice, then it was filtrated through a 0.2-mm membrane. The fluorescence intensity of the filtrate was determined by fluorescence spectrometer (FP-6500, Jasco, Japan).
WSFD, the cells were exposed to 32.4 J/cm2 visible light. After treatment, cells (1 106 cells per 3 mL in 6-well plates) were rinsed with D-Hanks solution and 10 lM DCF-DA was loaded. After 20 min incubation at 37 °C, the cells were harvested after being washed with PBS three times. The intracellular ROS accumulation was measured by using a Becton-Dickinson fluorescence-activated cell analyzer while data analysis was performed with Modifit LT 2.0 (Becton-Dickinson, San Jose, CA, USA). About 1 104 cells were counted for each analysis. The fluorescence intensity was quantified with CELLQuest software (Becton-Dickinson, Mountain View, CA, USA). 2.7. MTT reduction assay HeLa cells were plated at density of 1 104 cells per 100 lL in 96-well plates and the cell viability was determined by the conventional MTT reduction assay. After treatment, cells were treated with the 10 lL MTT solution (final concentration, 0.5 mg/mL) for 4 h. The dark blue formazan crystals formed in intact cells were solubilized with the lysis buffer (20% sodium dodecylsulfate in 50% aqueous N,N-dimethylformamide) and absorbance at 570/ 630 nm was measured with a microplate reader (Molecular Devices, Sunnylvale, CA, USA). 2.8. Determination of apoptosis Apoptotic cell death was analyzed by double staining with annexin V-FITC and propidium iodide (PI), in which annexin V bound to early apoptotic cells with exposed phosphatidylserine, while PI labeled the late apoptotic/necrotic cells with membrane damage. After 12 h incubation at 37 °C with WSFD, the cells were exposed to 32.4 J/cm2 visible light. After treatment, the apoptosis of cells was evaluated using an Annexin-V FITC apoptosis detection kit. Cells were harvested, washed and incubated at 4 °C for 30 min in the dark with annexin V-FITC and PI, then were analyzed on a FACS Vantage SE flow cytometer (Becton Dickinson, San Jose, CA, USA).
2.5. ROS detection in aqueous solutions 2.9. Determination of mitochondrial membrane potential The production of ROS was determined by measuring the intensity of green fluorescence emitted by redox-sensitive dyes dihydrorhodamine 123 (DHR; Sigma). After 32.4 J/cm2 visible light irradiation, the ROS generation in WSFD water mixture with DHR (5 mM) was assessed using fluorescence microplate reader (Chameleon; Hidex, Finland) equipped with a 488 nm excitation filter and a 535 nm emission filter. The results of ROS production by WSFD in water were presented as fold increase in DHR fluorescence in comparison with the control (water alone). The electron paramagnetic resonance (EPR) spectroscopy was used to monitor the generation of singlet oxygen in aqueous solutions. The EPR experiments were performed at room temperature on a Varian Eline spectrometer operating at a nominal frequency of 9.5 GHz. The aqueous solution containing 5 lg/mL of WSFD was supplemented with tetramethylpiperidine at 1 mM. Solutions were then exposed to illumination with visible light (32.4 J/cm2). Immediately after exposure to visible light, the sample-holding quartz tubes were transferred into the EPR cavity of the EPR spectrometer (Bruker BioSpin GmbH). Quantification of the signals was carried out by calculating the mean value of EPR signal amplitudes, and the data are expressed in arbitrary units. 2.6. Measurement of intracellular ROS accumulation The fluorescent probe DCF-DA was used to monitor the intracellular accumulation of ROS. After 12 h incubation at 37 °C with
HeLa cells were treated with WSFD for 12 h prior to visible light irradiation (32.4 J/cm2). After treatment, mitochondrial membrane potential (Dw) was assessed using DePsipher (R&D Systems), a lipophilic cation that had the property of aggregating upon membrane polarization forming an orange-red fluorescent compound. If the potential was disturbed, the dye cannot access the transmembrane space and remains or reverts to its green monomeric form. The cells were stained with DePsipher as described by the manufacturer, and the green monomer and the red aggregates were detected by flow cytometry. The results were presented as a red/green fluorescence ratio (FL2/FL1, arbitrarily set to 100% in untreated control samples), the increase and decrease of which reflect mitochondrial hyperpolarization and depolarization, respectively. 2.10. Measurement of caspase-3 activity HeLa cells were treated with WSFD for 12 h prior to visible light irradiation (32.4 J/cm2), and the caspase-3 activity was measured according to the manufacturer’s protocol. In brief, cells were lysed for 10 min in an ice bath, and centrifuged at 15,000g for 10 min at 4 °C, then the supernatant were incubated with acetyl-Asp-GluVal-Asp-aldehyde-AFC at 37 °C for 1 h. Fluorescence intensity was measured using a Jasco FP-6500 fluorescence spectrophotometer (kex 400 nm and kem 505 nm). The value for each treatment group was converted to the percentage of control.
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2.11. Measurement of MDA content and antioxidant enzyme activities For assay of MDA and antioxidant enzyme, the cultures were washed two times with phosphate-buffered saline (PBS), then scraped from the plates into ice cold PBS (0.1 M, containing 0.05 mM ethylene diamine tetraacetic acid) and homogenized. The homogenate was centrifuged at 4 °C at 10,000g for 30 min. The resulting supernatant was stored at 70 °C until the following analyses. Protein concentration was determined by the Bradford method, using bovine serum albumin as a reference standard. HeLa cells were treated with WSFD for 12 h prior to visible light irradiation (32.4 J/cm2). After 24 h incubation, the content of MDA was determined using the thiobarbituric acid method. Antioxidant enzyme activities were assayed after cells were incubated with WSFD for 12 h then exposed to 32.4 J/cm2 visible light. The processes of measurement were according to the instructions for the reagent kits. 2.12. Statistical analysis Values are reported as means ± S.D. Statistical comparisons were made by one-way ANOVA to detect significant difference using SPSS 13.0 for windows. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Characterization of WSFD In this work, amino group of amino acid and folic acid reacted with C60 to form the products. Analytic data of the synthesized and purified compounds were shown below. PFD: IR(KBr) m: 3421, 1709, 1604, 1496, 1068, 698, 579, 530 (C60 core), cm 1; 1H NMR(D2O) dppm: 7.21 (m), 4.02 (s), 3.43 (m), 2.84, 1.08 (m, C60–H); 13C NMR(D2O) dppm: 173.3, 138.7 (m, C60), 128.4, 57.5, 38.2; ESI-MS m/z (%): 1545 (M+, 34.6), 720 (100). Anal. calcd for C105H55N5O10: C 81.55, H 3.56, N 10.36; found C 80.87, H 3.87, N 10.56. The results indicated that there were five phenylalanine moieties per C60 molecule. FFD: IR(KBr) m: 3501, 3382, 2976, 1691, 1601, 1563, 1524, 1450, 1387, 1124, 1189, 570, 527 (C60 core), cm 1; 1H NMR(D2O) dppm: 9.0 (s), 7.7 (d), 6.8 (d), 4.3 (s), 3.6 (d), 2.3 (d), 2.1 (s), 1.9 (s), 1.2 (m, C60–H); 13C NMR(D2O) dppm: 174.9, 173.7, 161.1, 156.5, 150.7, 139.8–150.8 (m, C60), 118.6, 106.0, 55.6, 41.6, 31.2, 26.0; ESI-MS m/z (%): 1161 (M+, 38), 720 (100). Anal. calcd for C79H19N7O6: C 81.65, H 1.64, N 8.44; found C 79.76, H 1.87, N 8.93. The reaction on fullerene gave mono-adduct demonstrated by mass spectroscopy and elemental analysis. AFD: IR(KBr) m: 3430, 3187, 1638, 1574, 1401, 1190, 527 (C60 core), cm 1; 1H NMR(D2O) dppm: 3.58 (m), 2.84 (m), 2.02 (s),
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1.11 (m, C60–H); 13C NMR(D2O) dppm: 170.3, 137.0–155.0 (m, C60), 153.2, 77.0, 51.2, 39.8, 25.7, 23.6. ESI-MS m/z (%): 2112 (M+, 45.1), 720 (100). Anal. calcd for C108H112N32O16: C 61.36, H 5.30, N 21.21; found C 60.08, H 5.38, N 21.78. The results indicated that there were eight arginine moieties per C60 molecule of AFD. The intensity size distribution was measured by DLS particle-size analyzer (Fig. 2). The average hydrodynamic diameter of 1 mg/mL PFD, FFD and AFD was 5.5, 120.1 and 779.2 nm, respectively. 3.2. Assays of intracellular uptake of WSFD The fluorescence quantum yield of C60 was very low. Therefore, it was difficult to observe strong fluorescence of isolated C60 at normal experimental conditions. In aqueous solution of WSFD, interaction between adducts and C60 distorted the icosahedral symmetry of C60. The relaxation of selection rules due to the reduced symmetry of C60 molecule induced the observed strong fluorescence peaks [32]. The uptake of WSFD into HeLa cells was evaluated using a concentration of 5 lg/mL at different incubation times. In each case, the WSFD uptake was determined by fluorescence analysis. The results were summarized in Fig. 3A. WSFD was rapidly incorporated in the HeLa cells in the initial time (<4 h) and the uptake tended to a saturation value between 4 and 24 h. This value was estimated as 2.24, 2.98 and 0.85 lg per 106 cells for PFD, FFD and AFD, respectively. The result in our experiment demonstrated that WSFD could penetrate through the cell membrane to retain molecular stability, which further exhibited photodynamic activities in cytoplasm. The function groups with tumor targeting increased the uptake of WSFD in HeLa cells. In the same experiment condition, The N2a uptake of WSFD was estimated as 1.52, 1.37 and 0.41 lg per 106 cells for PFD, FFD and AFD, respectively. The uptake of WSFD in N2a cells was much less than HeLa cells (Fig. 3B). 3.3. ROS production by WSFD As it has previously been demonstrated that the cytotoxicity of WSFD was ROS mediated, we compared the ability of different WSFD to produce ROS. The data of DHR measurement of dosedependent ROS production in a cell free system (Fig. 4A) revealed a positive correlation between the cytotoxic capacity of the WSFD and their capacity to produce reactive oxygen. The EPR spectra of different WSFD showed that very strong TEMPOL signal was generated by PFD and FFD, while marginal signal was observed with AFD (Fig. 4B and C). The EPR data for the first time demonstrated a singlet oxygen production in water solutions of WSFD. However, it should be noted that the reactivity of non-specific ROS reporter dyes DHR indicated that other active oxygen species such as superoxide anion (O2 ), might also be generated in water solutions of WSFD. Accumulation of intracellular ROS was detected by using DCFDA. The cytotoxicity of WSFD towards HeLa cells was associated with an increase in the intracellular ROS production (Fig. 4D). When 5 lg/mL WSFD was irradiated by visible light, intracellular ROS accumulation was increased from 103.2 to 456.8, 512.9 and 198.6, respectively, which was determined by changes in fluorescence intensity. The same procedure without irradiation was carried out to determine the ROS generation in dark. The results of the intracellular ROS measurement, however, showed that FFD possessed the highest ROS-generating power. 3.4. WSFD-induced cytotoxicity and apoptosis in HeLa cells
Fig. 2. Size distribution functions of the colloidal dispersions formed by WSFD in water. The concentration of WSFD in the water was 1 mg/mL.
After 12 h incubation with WSFD followed by light irradiation, the phototoxicity of WSFD was tested by the MTT assay (Fig. 5A). The results showed that the decrease in cell viability was dependent on the irradiation time. The longer irradiation time, the higher
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Fig. 3. (A) Uptake of WSFD into HeLa cells as a function of incubation time. (B) Uptake of WSFD into N2a cells as a function of incubation time. The uptake of WSFD into cells was evaluated using a concentration of 5 lg/mL incubated for different incubation time. Values represent mean ± S.D. (n = 3).
Fig. 4. ROS production by WSFD. (A) Concentration-dependent ROS production by WSFD in water, measured as increase in DHR fluorescence. (B) The representative EPR spectra of 1O2 generation by WSFD. (C) EPR measurement of 1O2 generation by WSFD in comparison with control sample containing H2O. (D) Intracellular ROS generation by WSFD. The results from representative of at least three experiments are presented. Values represent mean ± S.D. (n = 3). ⁄p < 0.05 compared to the control group.
photodynamic inactivation of cell viability was found. The viability of cells pretreated with PFD, FFD and AFD at 5 lg/mL followed by 32.4 J/cm2 light irradiation was decreased to 44.2%, 27.4% and 76.4% of control value, respectively. The phototoxicity of WSFD presented the same trend as the light-induced ROS production, which confirmed the photodynamic effects of WSFD were resulted from their ability to generate ROS when photoexcited. Cell death was not observed when non-treated cell cultures were irradiated with visible light under the above described condition. Therefore, cell death of the cultures treated with the WSFD and then irradiated was caused by both visible light and the PS effect.
The next set of experiments was aimed at gaining some additional insight into the mechanisms of the phototoxic action of WSFD. We performed double staining of HeLa cells with annexin V-FITC and PI to detect phosphatidylserine exposure (annexin V) and cell membrane damage (PI). In control groups, 2.23% cells excluded PI and were positive for annexin V-FITC binding, which represented apoptotic cells. After 12 h incubation with 5 lg/mL WSFD followed by light irradiation (32.4 J/cm2), the percentage of apoptosis increased to 39.4%, 52.8% and 10.7%, respectively (Fig. 5B). In addition to apoptotic cells, the percentage of necrotic cells was 14.5%, 16.9% and 8.7% for PFD, FFD and AFD, respectively.
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Fig. 5. WSFD-induced cytotoxicity and apoptosis in HeLa cells. (A) HeLa cells were incubated with 5 lg/mL WSFD for 12 h prior to visible light irradiation (54 mW/cm2, different irradiated time). Cell viability was measured by the conventional MTT reduction assay. Data were presented as mean ± S.D. (n = 3). (B) HeLa cells were incubated with 5 lg/mL WSFD for 12 h prior to visible light irradiation (32.4 J/cm2). WSFD-induced apoptosis was determined by flow cytometry. Cells incubated with 5 lg/mL WSFD without irradiation served as dark group. Data were presented as mean ± S.D. (n = 3). ⁄p < 0.05 compared to the control group.
The results indicated that apoptotic cell death was predominant in all experiments. This was an advantageous result since apoptosis was an ordered process during an organism’s life cycle and took place without the drastic effect of inflammation as observed for necrosis. Our results also showed that 5 lg/mL WSFD didn’t induce dark cytotoxicity. 3.5. WSFD-induced apoptotic events The oxidation of membrane lipids, one of the primary events in oxidative cellular damage, can be assessed by measurement of MDA, a breakdown product of lipid peroxides. The cytotoxicity caused by ROS normally accompanied by the increase of lipid peroxides. Treatment of HeLa cells with WSFD followed by light irradiation caused significant increase in the intracellular MDA level (Table 1). PFD, FFD and AFD (5 lg/mL) increased the MDA level to 207.1%, 262.5% and 155.4% of control, respectively. The depolarization of mitochondrial membrane, an initial event in apoptosis induction, was readily detected after the light irradiation in the presence of WSFD, as demonstrated by a decrease in the red/green (FL2/FL1) fluorescence ratio of mitochondrial-binding dye DePsipher (Table 1). Treatment with WSFD followed by light irradiation significantly decreased the value of Dw. PFD, FFD and AFD (5 lg/mL) decreased the Dw to 44.6%, 34.8% and 81.4% of control, respectively. To further confirm the observation that WSFD induced apoptosis, caspase-3 activity was tested. Treatment with WSFD followed by irradiation significantly increased caspase-3 activity (Table 1), an enzyme family involved in apoptosis execution. Our results also showed that addition of WSFD without irradiation didn’t affect MDA level, Dw and caspase-3 activity (Table 2).
Table 1 Photodynamic effects of WSFD on MDA, mitochondrial membrane potential and caspase-3 activity in HeLa cells. Treatment
MDA (nmol/mg protein)
Dw (% of control)
Caspase-3 activity (% of control)
Control PFD FFD AFD
0.56 ± 0.08 1.16 ± 0.17* 1.47 ± 0.13 0.87 ± 0.09*
100.0 ± 5.1 44.6 ± 5.6* 34.8 ± 6.4* 81.4 ± 3.9*
100.0 ± 4.8 216.7 ± 10.8* 264.3 ± 8.9* 156.3 ± 11.6*
HeLa cells were incubated with WSFD (5 lg/mL) for 12 h prior to visible light irradiation (32.4 J/cm2). Data are presented as mean ± S.D. (n = 3). * p < 0.05 compared to the control group.
Table 2 Dark cytotoxicities of WSFD on MDA, mitochondrial membrane potential, caspase-3 activity and antioxidant enzyme activities in HeLa cells. Chemicals
PFD
FFD
AFD
MDA (nmol/mg protein) Dw (% of control) Caspase-3 (% of control) SOD (U/mg protein) CAT (U/mg protein) GSH-Px (U/mg protein)
0.54 ± 0.11 98.6 ± 3.2 104.6 ± 3.8 55.8 ± 3.7 26.7 ± 3.3 23.6 ± 3.8
0.52 ± 0.07 101.3 ± 4.9 105.3 ± 6.7 53.9 ± 4.3 26.3 ± 4.3 27.9 ± 3.9
0.55 ± 0.07 96.8 ± 5.3 102.9 ± 2.8 55.6 ± 5.9 25.4 ± 3.4 24.7 ± 2.7
HeLa cells were incubated with WSFD (5 lg/mL) for 36 h without visible light irradiation. Data are presented as mean ± S.D. (n = 3).
Table 3 Photodynamic effects of WSFD on antioxidant enzyme activities in HeLa cells. Treatment
SOD (U/mg protein)
CAT (U/mg protein)
GSH-Px (U/mg protein)
Control PFD FFD AFD
52.5 ± 4.1 16.3 ± 2.1* 9.8 ± 1.2* 30.8 ± 2.5*
23.6 ± 3.1 10.4 ± 0.7* 5.8 ± 0.7* 15.8 ± 2.1*
25.7 ± 2.6 8.9 ± 1.1* 4.8 ± 0.6* 16.1 ± 1.7*
HeLa cells were incubated with WSFD (5 lg/mL) for 12 h prior to visible light irradiation (32.4 J/cm2). Data are presented as mean ± S.D. (n = 3). * p < 0.05 compared to the control group.
3.6. WSFD caused loss of antioxidant enzyme activities in HeLa cells HeLa cells treated with WSFD followed by irradiation caused the decrease in the activity of SOD, CAT and GSH-Px (Table 3). Take FFD for example, cells treated with FFD and then irradiated caused the decrease in the activity of SOD, CAT and GSH-Px by 81.3%, 75.4% and 81.3%, respectively. Again, treatment with WSFD without irradiation didn’t alter the activities of SOD, CAT and GSH-Px in cultured HeLa cells (Table 2). 4. Discussion The potent ability of fullerene and its derivatives to photosensitize transition of molecular oxygen to highly reactive ROS makes them promising candidates for the photodynamic killing of cancer cells. The main advantage of this therapeutic approach is selectivity, achieved by tumor-specific activation of photosensitizing agent
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by highly focused light beam delivered to tumor region at the surface of the body or to internal tumors using optical fibers [33]. The effectiveness of various PS proposed for anticancer PDT can be judged on several criteria. The PS should be able to kill multiple classes of cancer cells at relatively low concentrations. PS should be reasonably non-toxic in the dark and should demonstrate selectivity for cancer cells over normal cells. The PS should actually be taken up inside the cell, and that the generation of ROS outside the cell will not be sufficient to produce efficient cell death. In present study, we have synthesized three WSFD, which have the potential to selectively kill cancer cells without dark toxicity. It was found that 5 lg/mL WSFD caused significant cell death after 10 min visible light irradiation. It should be noted that the photodynamic effect was apparently not restricted to HeLa cell line used in the present study, since it was also observed in other tumor cell lines, such as human laryngeal squamous cell carcinoma strain Hep-2 (Hu et al. unpublished observation). The clustering of WSFD in the aqueous phase, in which it was suggested to form micellar aggregate with the hydrophobic C60 core in the center and the hydrophilic groups were sticking into the water phase. The photodynamic effects of WSFD were associated primarily with conjugated molecular structure of C60. However, the morphology of the aggregate inhibited the photon energy absorbing and ROS generation of WSFD. The aggregation morphology of WSFD was mainly associated with the hydrophobic interaction and hydrogen bond. Both the chemical structure of adducts and addend numbers influenced the aggregate size of WSFD. In this work, the aggregate sizes of FFD and AFD were larger than PFD (AFD > FFD > PFD). It could be explained by considering the effect of hydrogen bond. FFD and AFD contained multiple amino and carboxylic groups within the same molecular framework, which self-assembled to form larger aggregates that were organized through bidentate hydrogen bonding interaction. However, the hydrophilic heads of PFD contained only one imino and one carboxylic group, which resulted in hydrogen bond hard to be formed between molecules. The hypothetic mechanism of the WSFD
self-aggregates was shown in Fig. 6. The data presented here on the PFD also showed that several hydrophilic addends were sufficient to prevent the strong hydrophobic three dimensional interactions among the fullerene moieties and the resulting tendency to form aggregates. Investigators demonstrated that both physical properties and chemical properties influenced the biological and biomedical activities of functionalized fullerenes [34]. The first factor contributing to radical-generation activity (i.e., 1O2 and O2 ) was the aggregate structure of WSFD. In present study, it was found that the ROS generation of WSFD in water (PFD > FFD > AFD) tended to decrease with increasing of aggregate sizes. In addition to chemical and physical properties of C60, other factors [35], such as cellular uptake, might profoundly influence the phototoxicity of C60-based agents. The results of intracellular ROS generation indicated by a disagreement of the ROS producing order (FFD > PFD > AFD) with the results got from ROS generation in cell free system (PFD > FFD > AFD). Since FFD possessed the highest uptake in HeLa cells, it could generate most ROS in cytoplasm. An important feature of various C60 preparations that facilitates their biological reactivity was the ability to penetrate cell membrane and gain access to cell cytoplasm, organelles and nucleus, as predicted by theoretical studies and confirmed in various experimental settings [36–38]. In present study, the WSFD uptake was determined by fluorescence analysis. The results indicated that the cellular uptake of WSFD was associated with functional groups. The proliferation rate of tumor cells is much faster than normal cells. Avid uptake of amino acids is a normal feature of rapidly proliferating cells. The membrane-associated folic acid receptor is a tumor marker that is overexpressed on a variety of neoplastic tissues, including breast, cervical, ovarian, colorectal, renal and nasopharyngeal tumors, but highly restricted in most normal tissues. Thus, both amino acids and folic acid can be used as targeting carrier for anticancer drugs. When amino acids and folic acid were covalently linked to C60 by amino moiety, its affinity for its cell surface receptor remained essentially unaltered. Further, following binding to
Fig. 6. The hypothetic mechanism of the WSFD self-aggregates in water.
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Fig. 7. The hypothetical mechanism of the photodynamic activities of WSFD. To overcome the poor solubility, biocompatibility and selectivity of C60, C60 was modified with Lphenylalanine, folic acid and L-arginine. As designed, the WSFD had good selectivity to tumor cells without dark cytotoxicity. After cell uptake and visible light irradiation, WSFD generated the 1O2 and other ROS (O2 ) in vitro. In parallel, following exposure of cells to WSFD and irradiation, a marked decrease in mitochondrial membrane potential, activities of SOD, CAT and GSH-Px were observed. Moreover, WSFD caused significant elevation in caspase-3 activity, and induced apoptotic death.
the cell surface receptor, the WSFD were internalized by cell. The sequence of the uptake of WSFD was FFD > PFD > AFD, which indicated folic acid had the best targeting among the three functional groups. Furthermore, the cellular uptake of WSFD in HeLa cells was much more than N2a cells, which indicated that WSFD had excellent selectivity to tumor cells. There two different mechanisms can explain the formation of cytotoxic species. Type I mechanism proceeds through formation of free radicals or O2 resulting from electron or hydrogen transfer. Type II mechanism is a process of the resonant energy transfer occurring during collisions between molecules of oxygen and sensitizer. In this process, the energy captured by a sensitizer is transferred to dioxygen, thus giving rise to generation of 1O2. The singlet excited state of C60 (1C60 ), initially formed upon light excitation, undergoes intersystem crossing to triplet state (3C60 ) that can be efficiently quenched by molecular oxygen to generate large amounts of 1O2. In the present study, the EPR data demonstrate the 1O2 production in water solutions of WSFD. This result indicates that the WSFD generates ROS by Type II mechanism. However, photoexcited C60 also efficiently mediates electron transfer from common electron donors (e.g., nicotinamide adenine dinucleotide) to electron acceptors such as oxygen to produce O2 . In the present study, the DHR assay data indicate that other active oxygen species such as O2 , might also be generated in water solutions of WSFD. This result indicates that the WSFD may also generate ROS by Type I mechanism. Thus, the WSFD generates ROS both by Type I and Type II mechanism in the photosensitization process. These unique photochemical properties have expanded WSFD applications into PDT. The oxidation of membrane lipids can be assessed by measurement of MDA. On the other hand, cells are often equipped with several antioxidant mechanisms (SOD, CAT and GSH-Px) that serve as a detoxifying system to prevent damage
caused by oxidative stress. The present study proved that cells treated with WSFD followed by light irradiation caused a marked decrease in cell survival, elevation of oxidative stress characterized by MTT and MDA production, and reduction in SOD, CAT and GSHPx activities. In addition to producing consequent lipid peroxidation and depolarization of mitochondrial membrane, WSFD exposure caused rising caspase-3 activity. Thus, the photodynamic effects of WSFD apparently involved induction of the ‘‘programmed’’ cell death, known as apoptosis [16]. This was consistent with the preferential mitochondrial localization of WSFD [36], having in mind that ROS induced mitochondrial dysfunction was a key initial step in the ‘‘mitochondrial’’ pathway of apoptosis [39]. The hypothetical mechanism was shown in Fig. 7. This study demonstrated that the photodynamic effects of fullerenes were linked to ROS generation under irradiation, which should be valuable for developing highly effective fullerene derivatives for use in biomedicine.
5. Conclusions We have synthesized three WSFD, which had tumor target photokilling activities without dark toxicity on HeLa cells. Both chemical properties and physical properties influenced the ROSgeneration abilities, cellular uptake and photodynamic activities of WSFD. The WSFD exerted photodynamic effects by targeted uptake and 1O2/O2 generation under irradiation with the following relative potencies: FFD > PFD > AFD. Pretreatment of the cells with WSFD followed by visible light irradiation induced cell apoptosis noticeably, such as oxidation of membrane lipids, depolarization of mitochondrial membrane, SOD, CAT and GSH-Px activities reduction and subsequent caspase-mediated apoptotic cell death.
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