An alternative 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 photosensitizer AlPcSs (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:sapphire os-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-section is the 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 μM AlPcS in serum-free medium was added
into the culture dish. The cells were incubated for 5 hours. These cells were 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
1
O2,
fluorescent
DBPF
changed
to
non-fluorescent
o-dibenzoylbenzene.2 The 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 μM AlPcS 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:
1
S. Sergeyev, O. Debever, E. Pouzet, and Y. H. Geerts, J. Mater. Chem. 17, 3002
(2007).
2
T. Ohyashiki, M. Nunomura, and T. Katoh, Biochim. Biophys. Acta. 1421, 131
(1999).