Abstract
The photosensitizing ability of C60/2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) nanoparticles under visible light irradiation was studied by electron spin resonance (ESR) and phototoxicity on cancer cells. In addition, the photoinduced antitumor effect to the tumor-bearing mice was evaluated. C60 nanoparticles were prepared by grinding a mixture of HP-β-CyD. The resulting C60/HP-β-CyD nanoparticles were highly-sensitive to visible light and generated higher levels of 1O2 than protoporphyrin IX (PpIX). C60/HP-β-CyD reduced the viability of cancer cells (HeLa cells and A549 cells) in response to irradiation by visible light in a dose-dependent manner. The IC50 values of the C60/HP-β-CyD nanoparticles was 10 μM for HeLa cells and 60 μM for A549 cells at an irradiation level of 35 mW/cm2. The photodynamic effect of C60/HP-β-CyD nanoparticles on the in vivo growth of mouse sarcoma S-180 cells was evaluated after intratumor injection. The outcome of PDT by C60/HP-β-CyD was directly dependent on the dose of irradiated light. Treatment with C60/HP-β-CyD nanoparticles at a C60 dose of 2.0 mg/kg under visible light irradiation at 350 mW/cm2 (63 J/cm2) markedly suppressed tumor growth, whereas that at 30 J/cm2 was less effective. These findings suggest that C60/HP-β-CyD nanoparticles represent a promising candidate for use in cancer treatment by PDT.
1. Introduction
Photodynamic therapy (PDT), which involves the production of reactive oxygen species (ROS), is a next-generation cancer treatment. In PDT, a photosensitizer is systemically administered, either locally or topically, and tumor sites are then irradiated by visible light to site-selectively generate ROS, leading to cell death and tissue destruction [1–3]. Therefore, PDT is an effective method for destroying diseased tissues without damaging surrounding healthy tissue. The ideal photosensitizer for use in PDT should meet the following requirements: (1) have high quantum yields for generating ROS, (2) have a strong absorbance with a high extinction coefficient in the long-wavelength (600~800 nm) region, where the maximum penetration of tissue by the penetrating light occurs [4], and (3) have minimal toxicity under conditions of nonlight irradiation. Many of the currently clinically employed photosensitizers are based on porphyrin molecules such as Photofrin, a hematoporphyrin derivative, and 5-aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX). However, these compounds have several disadvantages such as suboptimal tumor selectively and poor light penetration into tumors due to the absorption of the relatively short wavelengths. Therefore, more efficient photosensitizers are currently in various stages of development [5].
Fullerenes are currently of great interest for practical applications that take advantage of their unique electronic properties and biological activities [6–8]. The fullerene family, especially C60, is generally regarded as an efficient photosensitizer for PDT [9] due to light absorption at relatively long wavelengths and the high quantum yield of photoexcitation reactions [10, 11]. C60 molecules generate ROS such as singlet oxygen (1O2) and superoxide anion radicals () under light irradiation [10, 12]. In a previous study, we reported on the preparation of stable C60 nanoparticles (mean particle size ca. 90 nm), the surfaces of which were covered by hydrophilic 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) through physical adsorption and weak hydrophobic interactions [13]. The C60/HP-β-CyD nanoparticles showed cell toxicity due to their ability to generate ROS when irradiated with visible light [14]. However, few studies have focused on the in vivo photodynamic activity of C60. In spite of the advantages of PDT over conventional therapies, PDT is not extensively used, compared to the well-established chemo- and radiotherapy due to insufficient understanding of the procedures involved. In particular, the outcome of PDT is influenced by several factors such as the dose of the photosensitizer used, the presence of molecular oxygen, the irradiation time and the interval between drug administration and irradiation, and the correct dose of light. Thus, it is necessary to validate standard protocols for each individual photosensitizer in order to obtain an effective and reliable outcome [15]. In this study, we evaluated the photosensitizing ability of C60/HP-β-CyD nanoparticles under visible light irradiation as a new photosensitizer in PDT. In addition, we examined the in vivo photodynamic activity of C60/HP-β-CyD nanoparticles in tumor-bearing mice after visible light irradiation.
2. Materials and Methods
2.1. Materials
C60 (nanom purple SUH) was purchased from Frontier Carbon Co. (Tokyo, Japan). 2-Hydroxypropyl-β-CyD (HP-β-CD, degree of substitution (D.S.) of the 2-hydroxypropyl group was 5.6) was a gift from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Hydrophilic C60/HP-β-CyD nanoparticles were prepared by the coground method reported in previous paper [13]. ALA was purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). PpIX disodium salt was purchased from Sigma-Aldrich Co. LLC. (Tokyo, Japan) and dissolved in 10 mM NaOH or 10% dimethyl sulfoxide (DMSO) for ESR study and cell toxicity study, respectively. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was purchased from Labotec Co. Ltd. (Tokyo, Japan). 4-Hydroxy-2,2,6,6,-tetramethylpiperidine (TEMP-OH) was purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Eagle’s minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin-streptomycin were purchased from GIBCO Invitrogen Co. (Tokyo, Japan). Fetal calf serum (FCS) was obtained from Nichirei (Tokyo, Japan). All other materials and solvents were of analytical reagent grade and Milli-Q water was used throughout the study.
2.2. Photosensitizing Ability of C60/HP-β-CyD Nanoparticles under Visible Light Irradiation
The generation of and 1O2 from C60/HP-β-CyD nanoparticles and PpIX were measured using an X-band electron spin resonance (ESR) spectrometer (JES-FA100, JEOL Ltd., Tokyo, Japan) under the following conditions: microwave frequency 9.417 GHz, microwave power 4 mW, field modulation 0.1 mT at 100 kHz, and sweep time 30 s. was detected using DMPO as a spin-trapping reagent. The DMPO-OH signal was detected on ESR spectra, because the unstable DMPO-OOH adduct was rapidly converted to a DMPO-OH adduct. One hundred microliter aliquots of sample solutions and 80 μL of Milli-Q and 20 μL of DMPO were mixed well under aerobic conditions. 1O2 was also detected by the ESR using TEMP-OH as a spin-trapping reagent. One hundred microliters of a sample solution and 100 μL of TEMP-OH (160 mM) were well mixed under aerobic conditions. The mixed solutions were collected in a flat cell, then exposed to visible light generated by a xenon light source (MAX-303, Asahi spectra Co., Ltd., Tokyo, Japan), and subjected immediately to ESR measurement. Generation efficiency of and 1O2 was evaluated by the relative intensity to an external reference of Mn2+.
2.3. Photoinduced Cell Toxicity by C60/HP-β-CyD Nanoparticles
HeLa cells were cultured in MEM containing 100 units/mL penicillin-streptomycin, supplemented with 10% fetal bovine serum at 37°C and 5% of CO2. A549 cells were cultured in DMEM containing 100 units/mL penicillin-streptomycin, supplemented with 10% fetal bovine serum at 37°C and 5% of CO2. The cells were seeded in 96-well plates at a density of 2.0 × 104 cells/well for HeLa cells and 1.0 × 104 cells/well in the case of A549 cells. After growing overnight, the cells were incubated with the samples (1 μM~150 μM for C60 and 1 μM~300 μM for ALA) for 24 h in the dark. The cells were washed with phosphate-buffered saline (PBS) and replaced with fresh culture medium. The treated cells were exposed to light (35 mW/cm2, 400–700 nm) for 30 min, using a xenon light source. To measure the viability of cells as a ratio (%) compared with cells which were not treated with the samples, a WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay was carried out 24 h after the photoirradiation, by using a Cell Counting Kit (Dojindo Laboratories, Kumamoto, Japan). WST-1 works by reacting with the mitochondrial succinate-tetrazolium reductase forming the formazan dye. The effect of ROS scavengers on cell viability was studied used HeLa cells. The cells were incubated with C60/HP-β-CyD nanoparticles for 24 h and then washed with PBS and the medium was replaced with fresh medium including 10 mM of L-histidine, 2,5-dimethylfuran (DMF) and 1,4-diazabicyclo [] octane (DABCO) or 500 unit/mL of superoxide dismutase (SOD). After 1 h incubation, the treated cells were exposed to the light. To measure the viability of cells, WST-1 assay was carried out 24 h after the photoirradiation.
2.4. In Vivo Photodynamic Activity of C60/HP-β-CyD Nanoparticles
The care and maintenance of animals were conducted in accordance with the institutional guidelines of the Institutional Animal Care and Use Committee of Sojo University. To prepare a mouse tumor model, mouse sarcoma S-180 cells were acclimatized to the in vivo conditions by intraperitoneal growth in ddY mice (Kyudo Co. Ltd., Saga, Japan). The cells were implanted by subcutaneous injection in the dorsal skin of 6-week-old ddY mice (2 × 107 cells/mL, 100 μL/mouse). When the tumor mass reached a diameter of 7~8 mm, 0.1 mL of C60/HP-β-CyD nanoparticles in 5% glucose solution (C60 = 1 mM) were injected to the intratumor (2.0 mg/kg of C60, i.e., 0.072 mg of C60 per g body weight). The tumor tissue was exposed to visible light from a xenon light source at 400~700 nm (100~350 mW/cm2). Tumor volume (mm3) was calculated as /2 by measuring the length (L) and width (W) of the tumor on the dorsal skin.
3. Results and Discussion
3.1. Photosensitizing Ability of C60/HP-β-CyD Nanoparticles under Visible Light Irradiation
C60 is generally regarded to be an effective photosensitizer for PDT [9]; however, its extremely low solubility and poor dispersibility in water have significantly impeded pharmaceutical applications [16, 17]. Although several water soluble fullerene derivatives have been produced and their photodynamic activity evaluated [18, 19], only few studies in which the efficacy of C60 was compared with other photosensitizers have appeared. We therefore examined the in vitro photosensitizing ability of C60/HP-β-CyD nanoparticles, in comparison with that of PpIX by means of an ESR spin-trapping method. A xenon light source was used to produce homogenous light in the visible light range (Supplementary Figure 1(S) in Supplementary Material available online at http://dx.doi.org/10.1155/2014/570506). Figure 1(a) shows ESR spectra of C60/HP-β-CyD colloidal and PpIX solutions under photoirradiation. These photosensitizers produced three characteristic signals for TEMPO-OH in ESR spectra [20], indicating that 1O2 was generated by the photoirradiation. The relative intensity of the TEMPO-OH to an external reference (Mn2+) in these solutions increased with the photoirradiation time (Figure 1(b)). C60/HP-β-CyD nanoparticles generated higher levels of 1O2 than PpIX at every time period of photoirradiation. As shown in Figure 1(c), the generation of 1O2 from these photosensitizers was almost the same as that produced under low light energy (5 mW/cm2); however, the C60/HP-β-CyD nanoparticles were highly sensitive to the power of the light and large amounts of 1O2 were generated when a higher energy light source was used. It was estimated that at least 1.5 times larger energy is needed for PpIX to generate the same amount of 1O2 as that produced in the case of a C60 colloidal solution. For both C60/HP-β-CyD and PpIX, the generation of 1O2 under visible light irradiation was concentration dependent (Figure 1(d)). The generation of 1O2 at a low concentration (20 μM) was low in both the C60 and PplX systems, whereas C60/HP-β-CyD showed higher generation ability in the higher concentration range compared with that for PpIX. We previously reported that the HP-β-CyD-enhanced generation of 1O2 was due to the partial deposition of C60 in the hydrophobic CyD cavity [13], because the production of 1O2 shows a preference for nonpolar solvents. The low generation of 1O2 at low concentrations may be due to the dissociation of HP-β-CyD from the C60 nanoparticles. These collective results indicate that C60/HP-β-CyD has a potent photosensitizing ability when compared with PpIX under visible light irradiation. In addition, C60 has capability to generate when irradiated with visible light, as shown in Figure 2. A four-line ESR signal corresponding to DMPO-OH was detected in the ESR spectra [21], suggesting that is generated in the C60/HP-β-CyD colloidal solution. On the other hand, such a signal was not observed for PpIX. The generation of by C60/HP-β-CyD was also light power dependent and dose dependent, similar to that for the generation of 1O2 (Figure 1). C60/HP-β-CyD showed the higher absorption than that of PpIX at the region of 420 nm~540 nm and almost the same absorption spectra at the region of 600~700 nm where the maximum light absorption of C60 occurred at 620 nm, while that of PpIX was at 635 nm (Supplementary Figure 1(S)). The quantum yield for 1O2 at this wavelength was reported to be 0.96 for C60 in benzene [10], while the corresponding value was 0.56 for PpIX in phosphate buffer, the former value being higher than those for Photofrin and other photosensitizers [22, 23]. In this study, constant light energy was supplied in the wavelength range of 400~700 nm. Therefore, the different ROS generation ability of C60 and PpIX is probably due to not only the higher absorption at blue region but also the difference in the intrinsic photoexcitation properties of the photosensitizers. Further, it is known that extensive aggregation of photosensitizers significantly accelerates the decay of the excited triplet state [24], thus reducing the photosensitizing ability. In a previous study, we reported that C60 exists in the form of stable small aggregates with a particle size ca.90 nm in C60/HP-β-CyD colloidal solutions [13]. This might explain the higher photosensitizing ability of colloidal solutions of C60/HP-β-CyD, compared to PpIX solutions.
(a)
(b)
(c)
(d)
(a)
(b)
(c)
(d)
3.2. Photoinduced Cell Toxicity by C60/HP-β-CyDs Nanoparticles
The photodynamic activity of C60/HP-β-CyD nanoparticles on human cervical cancer HeLa cells and human lung carcinoma A549 cells was evaluated by WST-1 assays. We chose ALA as a control photosensitizer because PpIX is administered clinically in the form of a precursor. ALA is a naturally occurring amino acid that endogenously produced PpIX and the resulting PpIX can generate singlet oxygen leading to cancer cell death. The use of ALA has become a widely acceptable and popular procedure, particularly for the treatment of skin cancer [25]. As shown in Figure 3, C60/HP-β-CyD nanoparticles and ALA reduced the viability of these cells in response to the visible light irradiation and in a dose-dependent manner. The IC50 value of ALA for HeLa and A549 cells was 100 μM and 150 μM, respectively. In the case of C60/HP-β-CyD nanoparticles, the cellular survival significantly decreased at lower concentrations of C60 as a result of the photoirradiation. The IC50 value of the C60/HP-β-CyD nanoparticles was 10 μM for HeLa cells and 60 μM for A549 cells, which were much lower than those of ALA. This is probably due to the great ROS generation of C60/HP-β-CyD nanoparticles compared with ALA. When the cell viability test was conducted in the dark, no cell toxicity was observed at these concentrations of photosensitizers. In addition, C60/HP-β-CyD nanoparticles have capability to generate when irradiated with visible light as described above. In this study, the extracellular C60 was washed away prior to light irradiation; therefore, ROS such as and 1O2 might be generated from C60 taken up by cells, leading to the cell death. To assess the contribution of and 1O2 to the cell death, the effects of 1O2 scavengers, such as L-histidine, 2,5-dimethylfuran (DMF) and 1,4-diazabicyclo [] octane (DABCO) [20] and specific scavenger, superoxide dismutase (SOD), were studied. As shown in Figure 4, cell viability was increased by the addition of these ROS scavengers. These results indicate that both and 1O2 contribute to the potent photoinduced cell toxicity of C60/HP-β-CyD nanoparticles. However, we could not discuss which ROS is more effective to the cell death because the scavenging ability and cellular permeability of these scavengers were different from each other. Also, the amounts of C60 and ALA (or PpIX produced in the cells), especially C60, taken up in the cells could not be measured accurately due to the UV detection limit. The present results clearly indicate that C60/HP-β-CyD nanoparticles are toxic to cells only when irradiated by visible light, although further study is still needed to precisely evaluate the differences of the photosensitizers, C60 and ALA.
(a)
(b)
3.3. In Vivo Photodynamic Activity of C60/HP-β-CyD Nanoparticles
Since C60/HP-β-CyD nanoparticles demonstrated a potential to succeed as photosensitizer in PDT, a subsequent in vivo study was conducted to determine the standard protocols for the effective application of PDT by C60. Figure 5 shows the photodynamic effect of C60/HP-β-CyD nanoparticles on the in vivo growth of mouse sarcoma S-180 cells after intratumor injection. The tumor was irradiated for a short time period which was repeated at 30 s intervals. The tumor growth was similar to that observed for a saline injection, when treated with C60/HP-β-CyD nanoparticles at a C60 dose of 2.0 mg/kg, followed by visible light irradiation once at 100 mW/cm2 for 300 s (30 J/cm2) or 5 times at 200 mW/cm2 for 60 s (total light does 60 J/cm2). On the other hand, it was significantly suppressed when the irradiation power was increased, that is, 5 times at 250 mW/cm2 for 60 s (total light does 75 J/cm2). However, light irradiation alone at 75 J/cm2 significantly affected the tumor growth, as shown by the symbol of the empty triangle in Figure 5. In fact, the temperature increased to 50°C from 37°C when a mercury thermometer was directly light-irradiated at 250 W/cm2 for 60 s and normal skin was burned after being irradiated at this power. Therefore, it is very important to choose adequate irradiation doses to avoid damage to healthy tissues caused by a temperature increase. Figure 6 shows the photodynamic effects of C60/HP-β-CyD nanoparticles on tumor growth as the result of visible light irradiation 12 times at 350 mW/cm2 for 15 s (total light dose 63 J/cm2). As shown in Figures 6(a) and 6(b), tumor growth was significantly suppressed by this treatment at a dose of 2.0 mg/kg C60/HP-β-CyD. The treatment with C60/HP-β-CyD at a dose of 0.4 mg/kg was less effective against tumor growth, when compared with a dose of 2.0 mg/kg. The light irradiation alone had no effect on tumor growth and there was no significant difference compared with the saline injected group, suggesting a minor hyperthermia of the irradiation alone in this condition. The time profile for mice tumor growth treated with C60/HP-β-CyD nanoparticles without light irradiation was similar to that for the saline injection. Thus, C60/HP-β-CyD itself had no detectable cytocidal effect on the tumor tissue in the absence of irradiation. At this power, the temperature rise was within 5°C for 15 s and no light-induced skin damage was observed. It is interesting to note that the photodynamic effect was completely different depending on the protocol of light irradiation, even though the light energy supplied to tumor was almost the same (60 J/cm2 in Figure 5 and 63 J/cm2 in Figure 6). These results indicate that the PDT effect mainly depends on total light doses. Another parameter that limits tumor cell kill is the availability of oxygen in the tissue undergoing PDT treatment [26]. Since ROS arises from ground state oxygen, this process consumes oxygen in the tissue environment. The repeated intervals in the treatment at 63 J/cm2 might allow to oxygen to be regenerated and for a constant oxygen level to be maintained during PDT [27], leading to a sufficient PDT effect. Therefore, efficient PDT using C60 can be achieved by the repeated, short periods of high power irradiation. As shown in Figure 6(c), no significant difference in change of body weight was observed between C60/HP-β-CyD nanoparticles and the saline solution injected mice, suggesting that the C60/HP-β-CyD nanoparticles are relatively nontoxic. The light dose supplied in this experiment was not as high as the clinically used dose of 60~200 J/cm2 from a laser [15, 28]. Therefore, C60/HP-β-CyD nanoparticles appear to have great potential for use as a new photosensitizer for cancer treatment in PDT.
(a)
(b)
(c)
4. Conclusion
We investigated the potency of C60/HP-β-CyD nanoparticles as a new photosensitizer in the treatment of cancer. C60/HP-β-CyD nanoparticles had substantial photosensitizing ability compared with PpIX for the generation of 1O2 under visible light irradiation. In addition, C60/HP-β-CyD nanoparticles were capable of generating under visible light irradiation. The IC50 value of the C60/HP-β-CyD nanoparticles under photoirradiation was 10 μM for HeLa cells and 60 μM for A549 cells at an irradiation level of 35 mW/cm2. Efficient PDT using C60/HP-β-CyD nanoparticles in tumor-bearing mice was achieved by a treatment with C60/HP-β-CyD nanoparticles at a C60 dose of 2.0 mg/kg followed by 12 periods of visible light irradiation at 350 mW/cm2 for 15 s (total light dose 63 J/cm2). These results demonstrate that C60/HP-β-CyD nanoparticles are promising candidates for cancer treatment by PDT. The accumulation of C60 in the tumor and the pharmacokinetics of the process after injection of C60/HP-β-CyD nanoparticles are currently under investigation in our laboratory.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgments
This paper was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, under Grant no. 1-13-1432/HiCi. The authors, therefore, acknowledge with thanks DSR technical and financial support. This work was partly supported by JSPS KAKENHI Grant no. 23790062.
Supplementary Materials
(a) C60/HP-β-CyD shows the higher absorption than that of PpIX at the region of 420 nm~540 nm and almost the same absorption spectra at the region of 600~700 nm.
(b) Homogenous light is produced by a xenon light source in the visible light range.