Analternative model for photodynamic therapy of cancers: hot-band absorption

Jing Wang,Ji-Yao Chen*

State Key Laboratory of Surface Physics and Department of Physics, and Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Fudan University, Shanghai 200433, China.

Supplemental Materials:

Experimental Section

The sulfonated aluminum phthalocyanine (AlPcS), a popularly used PS in PDT, was synthesized as previously reported.1 The photosensitizerAlPcSs (Frontier Scientific, Inc) with the maximum four negative charges of the sulfonations on its four benzene rings (Figure 2A) can well dissolve in water. The absorption and fluorescence spectra of AlPcS were measured spectro-photometrically (Hitachi U-500 and Acton, Spectropro 2150i).

Our laser system comprises a Ti:sapphireos-cillator laser (Coherent Mira 900) which can be operated either in mode-unlocked (CW-) or in mode-locked regime. In our study, we use the first mode. We projected the CW laser beam onto a cuvette containing the dye sample solution. The induced fluorescence was directed onto the entrance slit of a spectrometer (Acton, Spectropro 2150i) through a multimode optical fiber. The fluorescence spectra was recorded on a liquid nitrogen-cooled CCD (Princeton, Spec-10:100B LN) that was mounted on the spectrometer. Because the populations of the vibration - rotation levels of the ground singlet state S0 of AlPcS molecules are determined by a Boltzmann distribution, the one-photon hot-band absorption cross-sectionisthe temperature dependent. We performed the whole experiment at room temperature (25 oC).

The KB cells (human nasopharyngeal carcinoma cell line) and the HeLa cells (human cervical carcinoma cell line) were obtained from the cell bank of Shanghai Science Academy. The cells were seeded in culture dishes containing dulbecco modified eagle medium (DMEM) and 10% calf serum, 100 units ml−1 penicillin, 100 μg ml−1 streptomycin and 100 μg ml−1 neomycin. They were incubated in a fully humidified incubator at 37 oC with 5% of CO2. Cells in the exponential phase of growth were used in our experiments. 20 μMAlPcS in serum-free medium was added into the culture dish. The cells were incubated for 5 hours. These cellswere then washed three times with phosphate buffered solution (PBS) to remove unbound AlPcS for the further experiments.

1,3-Diphenylisobenzofuran (DPBF), a sensitive probe for singlet oxygen (1O2), was used to detect the 1O2 produced in AlPcS photosensitization. Upon oxidative degradation by 1O2, fluorescent DBPF changed to non-fluorescent o-dibenzoylbenzene.2The fluorescence of DPBF under the 405 nm excitation was measured in a spectrometer (Acton, Spectropro 2150i and Hitachi, F-2500). In the experiment, DPBF (18 μM) mixed with AlPcSs (20 μM) in aqueous solutions were irradiated by the 800 nm CW laser (280 mW). The DPBF photo-degradation was measured to deduce the 1O2 production. For comparison, conventional 1O2 production induced by the red light (7 mW) was also studied. A red light beam (630 - 710 nm) filtered from a halogen lamp projector was used as the light source.

A new kind of MTT kit, the Cell Proliferation and Cytotoxicity Assay Kit (WST-1), was used to measure the killing effect of AlPcS on two cancer cell lines. The cells (either HeLa cells or KB cells) were cultured in culture dishes (35 mm diameter). When the cells reached 80% confluence with normal morphology, 20 μMAlPcS in serum-free culture medium was added into the culture dish and incubated for 5 hours. After incubation, the cells were washed with PBS three times to remove the unassociated compound, and added with fresh medium. These cells were then digested by pancreatin for 2 minutes. The cell suspension was collected in a sterile glass tube and was irradiated by either the CW 800 nm laser (280 mW) or a 630 -710 nm light beam (5 mW). After irradiation, the cells were seeded in wells of a 96-well flat bottom tissue culture plate with 104 cells per well. Cells in the well plate were incubated for 24 h. 10 μl MTT solution (5 mg/ml) was then added into each well for incubation of 1.5 h. Finally, the optical densities (O.D) at the wavelength of 450nm for each well were measured on an iEMS Analyzer (Lab-system). The cell viability in each well was determined by comparing the O.D value with that of untreated control cells in wells of the same plate. All results were presented as the mean ± SE from three independent experiments with four wells in each.

REFERENCES:

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2T.Ohyashiki, M.Nunomura, and T.Katoh,Biochim.Biophys.Acta.1421, 131 (1999).