alone resulted in slight cytotoxicity (22%). Laser irradiation alone (without βCD-4TFEO-Pc) resulted in virtually no cytotoxicity. In cells treated with a combination of βCD-4TFEO-Pc and laser irradiation, tumor growth was inhibited significantly by laser irradiation in a dose-dependent manner. This dependency of the dose of laser irradiation was also confirmed in the case of other zinc Pc derivatives and cell lines (data not shown).
3.3. In Vivo Antitumor Activity Using Chick Embryos
βCD-4TFEO-Pc showed excellent antitumor activity in vitro; therefore, photosensitizers were evaluated in an in vivo antitumor assay system using fertilized chicken eggs. Tumor cells of various species can be inoculated on the CAM because chicken eggs are naturally immunodeficient before hatching, and good proliferation of tumor cells has been observed in several studies  -  . Several investigations using the chick embryo assay to evaluate antitumor activity in vivo have been carried out. This assay method is rapid, convenient, inexpensive, and causes less pain to experimental animals. The antitumor effects of βCD-4TFEO-Pc against B16-F10 cells transplanted onto the CAM of a chick embryo are shown in Figure 4. The tumor IR of βCD-4TFEO-Pc with laser irradiation was 52.7% (p < 0.001), and that of laser irradiation alone and photosensitizer alone was <7% at 50 µg per egg and at 100 J/cm2 (150 mW).
Zinc Pc derivatives are expected to be second-generation photosensitizers because they have relatively high absorbance at red light, which can reach deep into tissues. However, they show π-π stacking, which results in aggregation in water, and they also have low solubility   . To overcome these drawbacks, we synthesized novel zinc Pc derivatives containing fluorine    and also conjugate CD molecules.
In novel zinc Pc derivatives, βCD-4TFEO-Pc exhibited greater photodynamic effects than those seen with talaporfin, which is used widely for the treatment of early-stage lung cancer and malignant primary brain tumors. Our study showed that βCD-4TFEO-Pc was non-toxic and that laser irradiation was needed for βCD-4TFEO-Pc to exhibit its antitumor activity because it can produce singlet oxygen as an active species to abrogate tumor proliferation. Though the wavelength used (664 nm) was not optimal against βCD-4TFEO-Pc, it showed great sensitivity to laser irradiation. The equipment to create laser light was developed for the PDT of talaporfin in clinical scenarios; therefore, the wavelength of irradiation matched exactly to the intense absorption of talaporfin
Figure 3. Correlation of laser energy and cytotoxicity of B16-F10 melanoma- treated β-CD-4TFEO-Pc in vitro. B16-F10 cells were treated with photosensitizer at various concentrations for 5 h. After removing the photosensitizer, cells were irradiated with or without the laser (0 (■), 25 (△), 50 (▲), 100 (○), 200 (●) J/cm2, 664 nm). Cytotoxicity was determined by the MTT assay 24 h after irradiation. Each point is the mean of ≥3 independent experiments, and error bar represents the standard deviation.
Figure 4. In vivo antitumor activity of βCD-4TFEO-P determined using a chick embryo assay. Antitumor activity of βCD-4TFEO-Pc with laser irradiation against the growth of B16-F10 tumor cells on CAM was evaluated on day 17. aSignificance determined by the Student’s t-test (p < 0.001) is indicated. Values show the inhibition ratio (%) vs. control tumor weight.
 . The intense absorption of βCD-4TFEO-Pc was shifted to around 700 nm owing to modification of the Pc skeleton, and light of wavelength 664 nm that was irradiated was not of sufficient wavelength to exert the maximum photodynamic effect for βCD-4TFEO-Pc. Nevertheless, the excellent antitumor activity of βCD-4TFEO- Pc was demonstrated in the present study and its clinical application as a photosensitizer is expected. A photosensitizer that is sensitive to a specific light source and laser irradiation are important factors in PDT for treatment of superficial cancer. The dependency of laser irradiation is revealed in Figure 3 and it was suggested that a high-powered laser exhibited antitumor activity. High-power laser irradiation can produce heat at localized irradiated areas; thus, evaluation of antitumor activity is thought to involve photodynamic and hyperthermic effects. At 100 J/cm2, laser irradiation showed effective antitumor effects without producing heat. Even low-power laser irradiation could activate photosensitizers to produce singlet oxygen as an active species to abrogate tumor proliferation.
Conjugation of CDs to zinc Pc dramatically improved water solubility, and coating of fluorine to zinc Pc prevented self-aggregation. When several zinc Pc derivatives coated with fluorine (including 4TFEO-Pc) were examined for photodynamic effects, it appeared that introduction of fluorine led to an increase in photodynamic effects. CD conjugation resulted in an increase in hydrophilicity (which is thought to be a useful property for intestinal absorption, increases in bioavailability, and drug preparation)   . Conventional photosensitizers exhibit non-specific cytotoxicity against non-irradiated cells; hence, patients should shade from light including sunbeam such treatment for several days to avoid side effects. Even high concentrations of βCD-4TFEO-Pc showed very low cytotoxicity upon treatment with βCD-4TFEO-Pc alone. Low sensitivity is a very useful property for a photosensitizer. This useful phenomenon was observed with derivatives with fluorine and CD conjugation. βCD comprising six glucose molecules seemed to be optimal for photodynamic effects; thus, the Pc motif probably matched the pore size of βCD.
The mother compound, Zinc Pc, showed higher cytotoxicity than βCD-4TFEO-Pc and talaporfin in B16-F10 and HT-1080 cells. This activity was non-specific for laser irradiation in B16-F10 cells. Laser irradiation did not completely affect the chemosensitivity of zinc Pc. It seems that the extremely high cytotoxicity of zinc Pc masked photodynamic effects. However, in HT-1080 cells, zinc Pc showed moderate photodynamic effects with high cytotoxicity. Thus, we suspected that laser irradiation was not sufficient to penetrate B16-F10 cells (which originate from melanoma and contain large amounts of melanin pigment), because there were reported that pigmented tumors were resistance to the PDT   . B16-F10 and HT-1080 cells treated with talaporfin and laser irradiation achieved weak photodynamic effects, but at high doses, the photodynamic effect elicited by talaporfin disappeared. The range of drug concentrations used was narrow; therefore, the plasma concentration after drug administration and area of laser irradiation should be monitored very carefully.
In this paper, the CAM assay using chicken eggs as in vivo chemosensitivity test was performed. The model of chick embryos can correctly predict a clinical response of chemotherapy in the several tumors such as lung cancer and malignant glioma and any study as in vivo model were adapted the CAM assay    . A total of 50 µg/egg βCD-4TFEO-Pc was administered into the CAM vein of eggs and the inhibitory effect of tumor growth was evaluated by tumor weight on day 17 from fertilization. A significant difference in the βCD-4TFEO- Pc group plus laser irradiation compared with the control group was noted with regard to tumor proliferation on the CAM of eggs, but laser irradiation and βCD-4TFEO-Pc alone did not show inhibitory effects. Moreover, in one egg with βCD-4TFEO-Pc plus laser irradiation, the implanted tumor was quite small on day 17. If an optimal dose and timing of βCD-4TFEO-Pc administration and optimal wavelength of laser irradiation are applied, excellent antitumor activity (e.g., abrogation of tumor proliferation) could be achieved.
We suggest that βCD-4TFEO-Pc is a useful photosensitizer for the treatment of superficial cancers. If a high- power light source  to match its specific wavelength can be developed, excellent treatment of superficial cancers could be achieved by applying βCD-4TFEO-Pc for PDT.
The authors thank the Pharmaceutical Research Center of Meiji Seika Pharma and Panasonic. The in vivo antitumor experiment performed using chick embryo was approved (13-023) by the ethics committee of Aichi Gakuin University.