Potential Use of C 60 / 2-Hydroxypropyl-β-cyclodextrin Nanoparticles as a New Photosensitizer in the Treatment of Cancer

The photosensitizing ability of C 60 /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. C 60 nanoparticles were prepared by grinding a mixture of HP-β-CyD. The resulting C 60 /HPβ-CyD nanoparticles were highly-sensitive to visible light and generated higher levels of O 2 than protoporphyrin IX (PpIX). C 60 /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 IC 50 values of the C 60 /HP-β-CyD nanoparticles was 10 μM for HeLa cells and 60 μM for A549 cells at an irradiation level of 35mW/cm. The photodynamic effect of C 60 /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 C 60 /HP-β-CyD was directly dependent on the dose of irradiated light. Treatment with C 60 /HP-β-CyD nanoparticles at a C 60 dose of 2.0mg/kg under visible light irradiation at 350mW/cm (63 J/cm) markedly suppressed tumor growth, whereas that at 30 J/cm was less effective. These findings suggest that C 60 /HP-β-CyD nanoparticles represent a promising candidate for use in cancer treatment by PDT.


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][2][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 5aminolevulinic 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][7][8].The fullerene family, especially C 60 , 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].C 60 molecules generate ROS such as singlet oxygen ( 1 O 2 ) and superoxide anion radicals (O 2 •− ) under light irradiation [10,12].In a previous study, we reported on the preparation of stable C 60 nanoparticles (mean particle size ca.90 nm), the surfaces of which were covered by hydrophilic 2-hydroxypropyl--cyclodextrin (HP--CyD) through physical adsorption and weak hydrophobic interactions [13].The C 60 /HP--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 C 60 .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 C 60 /HP--CyD nanoparticles under visible light irradiation as a new photosensitizer in PDT.In addition, we examined the in vivo photodynamic activity of C 60 /HP--CyD nanoparticles in tumor-bearing mice after visible light irradiation.

Materials and Methods
2.1.Materials.C 60 (nanom purple SUH) was purchased from Frontier Carbon Co. (Tokyo, Japan).2-Hydroxypropyl--CyD (HP--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 C 60 /HP--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.•− 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 L of Milli-Q and 20 L of DMPO were mixed well under aerobic conditions. 1 O 2 was also detected by the ESR using TEMP-OH as a spin-trapping reagent.One hundred microliters of a sample solution and 100 L 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 O 2

Photosensitizing Ability of C
•− and 1 O 2 was evaluated by the relative intensity to an external reference of Mn 2+ .

Photoinduced Cell
Toxicity by C 60 /HP--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 CO 2 .A549 cells were cultured in DMEM containing 100 units/mL penicillinstreptomycin, supplemented with 10% fetal bovine serum at 37 ∘ C and 5% of CO 2 .The cells were seeded in 96-well plates at a density of 2.0 × 10 4 cells/well for HeLa cells and 1.0 × 10 4 cells/well in the case of A549 cells.After growing overnight, the cells were incubated with the samples (1 M∼ 150 M for C 60 and 1 M∼300 M 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/cm 2 , 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 C 60 /HP--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 [2, 2, 2] 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.

In Vivo Photodynamic Activity of C 60 /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 × 10 7 cells/mL, 100 L/mouse).When the tumor mass reached a diameter of 7∼8 mm, 0.1 mL of C 60 /HP--CyD nanoparticles in 5% glucose solution (C 60 = 1 mM) were injected to the intratumor (2.0 mg/kg of C 60 , i.e., 0.072 mg of C 60 per 35 ± 2 g body weight).The tumor tissue was exposed to visible light from a xenon light source at 400∼700 nm (100∼350 mW/cm 2 ).Tumor volume (mm 3 ) was calculated as ( 2 × )/2 by measuring the length (L) and width (W) of the tumor on the dorsal skin.

Photosensitizing Ability of C 60 /HP-𝛽-CyD Nanoparticles
under Visible Light Irradiation.C 60 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 C 60 was compared with other photosensitizers have appeared.We therefore examined the in vitro photosensitizing ability of C 60 /HP--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 C 60 /HP--CyD colloidal and PpIX solutions under photoirradiation.These photosensitizers produced three characteristic signals for TEMPO-OH in ESR spectra [20], indicating that 1 O 2 was generated by the photoirradiation.The relative intensity of the TEMPO-OH to an external reference (Mn 2+ ) in these solutions increased with the photoirradiation time (Figure 1(b)).C 60 /HP--CyD nanoparticles generated higher levels of 1 O 2 than PpIX at every time period of photoirradiation.As shown in Figure 1(c), the generation of 1 O 2 from these photosensitizers was almost the same as that produced under low light energy (5 mW/cm 2 ); however, the C 60 /HP--CyD nanoparticles were highly sensitive to the power of the light and large amounts of 1 O 2 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 1 O 2 as that produced in the case of a C 60 colloidal solution.For both C 60 /HP--CyD and PpIX, the generation of 1 O 2 under visible light irradiation was concentration dependent (Figure 1(d)).The generation of 1 O 2 at a low concentration (20 M) was low in both the C 60 and PplX systems, whereas C 60 /HP--CyD showed higher generation ability in the higher concentration range compared with that for PpIX.We previously reported that the HP--CyDenhanced generation of 1 O 2 was due to the partial deposition of C 60 in the hydrophobic CyD cavity [13], because the production of 1 O 2 shows a preference for nonpolar solvents.The low generation of 1 O 2 at low concentrations may be due to the dissociation of HP--CyD from the C 60 nanoparticles.These collective results indicate that C 60 /HP--CyD has a potent photosensitizing ability when compared with PpIX under visible light irradiation.In addition, C 60 has capability to generate O 2 •− 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 O 2 •− is generated in the C 60 /HP--CyD colloidal solution.On the other hand, such a signal was not observed for PpIX.The generation of O 2 •− by C 60 /HP--CyD was also light power dependent and dose dependent, similar to that for the generation of 1 O 2 (Figure 1).C 60 /HP--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 C 60 occurred at 620 nm, while that of PpIX was at 635 nm (Supplementary Figure 1(S)).The quantum yield for 1 O 2 at this wavelength was reported to be 0.96 for C 60 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 C 60 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 C 60 exists in the form of stable small aggregates with a particle size ca.90 nm in C 60 /HP--CyD colloidal solutions [13].This might explain the higher photosensitizing ability of colloidal solutions of C 60 /HP--CyD, compared to PpIX solutions.

Photoinduced Cell Toxicity by C 60 /HP-𝛽-CyDs Nanoparticles.
The photodynamic activity of C 60 /HP--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, C 60 /HP--CyD nanoparticles and ALA reduced the viability of these cells in response to the visible light irradiation and in a dose-dependent manner.The IC 50 value of ALA for HeLa and A549 cells was 100 M and 150 M, respectively.In the case of C 60 /HP--CyD nanoparticles, the cellular survival significantly decreased at lower concentrations of C 60 as a result of the photoirradiation.The IC 50 value of the C 60 /HP--CyD nanoparticles was 10 M for HeLa cells and 60 M for A549 cells, which were much lower than those of ALA.This is probably due to the great ROS generation of C 60 /HP--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, C 60 /HP--CyD nanoparticles have capability to generate O 2 •− when irradiated with visible light as described above.In this study, the extracellular C 60 was washed away prior to light irradiation; therefore, ROS such as O 2 •− and 1 O 2 might be generated from C 60 taken up by cells, leading to the cell death.To assess the contribution of O 2 •− and 1 O 2 to the cell death, the effects of 1 O 2 scavengers, such as L-histidine, 2,5-dimethylfuran (DMF) and 1,4-diazabicyclo [2, 2, 2] octane (DABCO) [20] and O 2 •− 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 O 2 •− and 1 O 2 contribute to the potent photoinduced cell toxicity of C 60 /HP--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 C 60 and ALA (or PpIX produced in the cells), especially C 60 , taken up in the cells could not be measured accurately due to the UV detection limit.The present results clearly indicate that C 60 /HP--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, C 60 and ALA.protocols for the effective application of PDT by C 60 .Figure 5 shows the photodynamic effect of C 60 /HP--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 C 60 /HP--CyD nanoparticles at a C 60 dose of 2.0 mg/kg, followed by visible light irradiation once at 100 mW/cm 2 for 300 s (30 J/cm 2 ) or 5 times at 200 mW/cm 2 for 60 s (total light does 60 J/cm 2 ).On the other hand, it was significantly suppressed when the irradiation power was increased, that is, 5 times at 250 mW/cm 2 for 60 s (total light does 75 J/cm 2 ).However, light irradiation alone at 75 J/cm 2 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/cm 2 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 C 60 /HP--CyD nanoparticles on tumor growth as the result of visible light irradiation 12 times at 350 mW/cm 2 for 15 s (total light dose 63 J/cm 2 ).As shown in Figures 6(a 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/cm 2 in Figure 5 and 63 J/cm 2 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/cm 2 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 C 60 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 C 60 /HP--CyD nanoparticles and the saline solution injected mice, suggesting that the C 60 /HP--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/cm 2 from a laser [15,28].Therefore, C 60 /HP--CyD nanoparticles appear to have great potential for use as a new photosensitizer for cancer treatment in PDT.

Conclusion
We investigated the potency of C 60 /HP--CyD nanoparticles as a new photosensitizer in the treatment of cancer.C 60 /HP--CyD nanoparticles had substantial photosensitizing ability compared with PpIX for the generation of 1 O 2 under visible light irradiation.In addition, C 60 /HP--CyD nanoparticles were capable of generating O 2 •− under visible light irradiation.The IC 50 value of the C 60 /HP--CyD nanoparticles under photoirradiation was 10 M for HeLa cells and 60 M for A549 cells at an irradiation level of 35 mW/cm 2 .Efficient PDT using C 60 /HP--CyD nanoparticles in tumorbearing mice was achieved by a treatment with C 60 /HP--CyD nanoparticles at a C 60 dose of 2.0 mg/kg followed by 12 periods of visible light irradiation at 350 mW/cm 2 for 15 s (total light dose 63 J/cm 2 ).These results demonstrate that C 60 /HP--CyD nanoparticles are promising candidates for cancer treatment by PDT.The accumulation of C 60 in the tumor and the pharmacokinetics of the process after injection of C 60 /HP--CyD nanoparticles are currently under investigation in our laboratory.

Figure 1 :
Figure 1: ESR spectra of the TEMPO-OH adduct generated in a C 60 /HP--CyD colloidal solution and a PpIX solution (C 60 and PpIX = 80 M, light irradiation at 25 mW/cm 2 for 5 min) (a).Its relative intensity increment after visible light irradiation.(b) Effects of the light power (c) and concentrations of photosensitizers (d) on the generation of 1 O 2 .Empty circle: C 60 /HP--CyD nanoparticles; empty square: PpIX.Each point represents the mean ± S.E. of 3 experiments.*  < 0.05 versus PpIX.

Figure 2 :
Figure 2: ESR spectra of the DMPO-OH adduct generated in the C 60 /HP--CyD colloidal solution (C 60 = 40 M, light irradiation at 40 mW/cm 2 for 5 min) (a).Its relative intensity increment after visible light irradiation (b).Effects of the light power (c) and concentrations of photosensitizers (d) on the generation of O 2 •− .Each point represents the mean ± S.E. of 3 experiments.

Figure 3 :Figure 4 :
Figure 3: Photodynamic activities of C 60 /HP--CyD nanoparticles and ALA for HeLa cells (a) and A549 cells (b) after visible light irradiation at 35 mW/cm 2 .Black filled circle: C 60 /HP--CyD in the dark; black filled triangle: ALA in the dark; empty circle: C 60 /HP--CyD with light irradiation; empty triangle: ALA with light irradiation.Each value points the mean ± S.E. of 3-5 experiments.*  < 0.05 versus ALA with light irradiation.