Cu(ii)-BODIPY photosensitizer for CAIX overexpressed cancer stem cell therapy

Chemoresistance originating from cancer stem cells (CSCs) is a major cause of cancer treatment failure and highlights the need to develop CSC-targeting therapies. Although enormous progress in both photodynamic therapy (PDT) and chemodynamic therapy (CDT) has been made in recent decades, the efficacy of these modalities against CSC remains limited. Here, we report a new generation photosensitizer, CA9-BPS-Cu(ii), a system that combines three subunits within a single molecule, namely a copper catalyst for CDT, a boron dipyrromethene photosensitizer for PDT, and acetazolamide for CSC targeting via carbonic anhydrase-9 (CA9) binding. A therapeutic effect in MDA-MB-231 cells was observed that is ascribed to elevated oxidative stress mediated by a combined CDT/PDT effect, as well as through copper-catalysed glutathione oxidation. The CSC targeting ability of CA9-BPS-Cu(ii) was evident from the enhanced affinity of CA9-BPS-Cu(ii) towards CD133-positive MDA-MB-231 cells where CA9 is overexpressed vs. CD133-negative cells. Moreover, the efficacy of CA9-BPS-Cu(ii) was successfully demonstrated in a xenograft mouse tumour model.


Spectroscopic data
For UV/Vis and fluorescence spectra measurements, a dimethyl sulfoxide (DMSO) stock solution of CA9-BPS-Cu(II) and CA9-BPS was diluted with aqueous buffer (10 mM PBS, pH 7.4, 5% DMSO) or ethanol, to give a 5 μM solution. For fluorescence spectral measurements, samples were excited at 660 nm using excitation and emission slit widths of 20 nm.

Fluorescence quantum yield (Φf) calculations
Fluorescence quantum yields were determined by comparing the fluorescence intensity of the sample (PS: photosensitizer) with that of a fluorescence standard (R: reference) using the following equation: -----Eqn. S1 Φf is the quantum yield, A is the integrated area under the corrected fluorescence spectra, OD is the optical density, and n is the refractive index. The subscript PS and R refer to the sample and reference, respectively. Zinc phthalocyanine, for which the fluorescence quantum yield (Φf) in toluene containing 1.0 % pyridine is 0.30, was used as the reference. S4

Analyses of singlet oxygen generation
The generation of singlet oxygen was measured by mixing 80 μM 1,3-diphenylisobenzofuran (DPBF) and 1 μM PS in acetonitrile with adjusting the absorbance of DPBF and PS to around 1.0 and 0.1, respectively. In the case of studying the effect of thiol, Na2S (1eq) was pre-S7 incubated with CA9-BPS-Cu(II). After measuring the UV-Vis spectrum in the dark, the samples were exposed to monochromatic light at 660 nm (Xenon Lamp Monochromatic Light Source, slit width 15/1.5 nm) for 3 minutes. After each irradiation, UV-Vis spectra were collected. The absorbance of DPBF at 412 nm was plotted versus time and the slope was determined.

Generation of knockout cell lines with CRISPR-Cas9 system
The CRISPR-Cas9 system was prepared as follows: The CA9-guide-RNA targeting of the CA9 hours before being replaced with complete medium for the desired duration. One days after transfection, cells were diluted 96 well plate for single colony formation. After 10 days, colonies were isolated and analyzed by western blotting for transfection efficiency.

Cytotoxicity analyses
To determine the cytotoxicity, a Cellomax Cell Viability Kit (Precaregene, Hanam, Gyeonggi- Kensington, PA, USA). All cell-seeded plates were prepared in triplicate to allow comparisons between normoxic and hypoxic effects (21% vs. 3% O2). All culture media and phosphatebuffered saline (PBS) were pre-incubated overnight in a standard 5% CO2 incubator so as to be pre-gassed after purging with pure nitrogen to remove dissolved O2 before being subject to further incubation in a 3% oxygen environment until the dissolved O2 levels equilibrated to 3%.
Cells were incubated at 37 °C for an additional 6 h and then washed with PBS twice. The cells were then irradiated using a 660 nm LED lamp (100 mW/cm 2 , 5 min; 30 J/cm 2 ; DavinchK, Seoul, Korea). The absorbance of the wells was detected at 450 nm by a Hidex Sense microplate reader (Hidex, Cranbourne, Victoria, AU). Cell viability assays were performed in triplicate and the cytotoxicity was recorded as a percentage calculated for the treated cells relative to the control group.

Combination index (CI) analyses
To assess the synergy of the chemodynamic therapy (CDT) and photodynamic therapy (PDT), the so-called combination index (CI) used to quantify synergism or antagonism for two drugs was employed as follows: S8 where CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. In the denominators, (Dx)1 represents D1 "alone" that inhibits a system x%, and (Dx)2 is for D2 "alone" that inhibits a system x%. In the numerators, (D)1 and (D)2 "in combination" also inhibit x%. CI was calculated for every dose of two drug pairs. Fraction affected (Fa) is the fractional inhibition of a phenotype by a compound treatment(s). Fa of a group was calculated S11 as Fa = percent inhibition of cell viability/100. Fraction affected-combination index plot was drawn with every group treated with more than one compound.

Intracellular ROS measurements
Cells were seeded on a confocal dish (2.0 × 10 5 per dish) and cultured for a day. The cells were then washed with PBS twice before fresh culture medium containing CA9-BPS-Cu(II) or

Intracellular thiol detection
Cells were seeded on a confocal dish (2.0 × 10 6 per dish), and incubated for a day.

Tumor spheroids formation and PI cytotoxicity
In

Immunocytochemistry of cryosectioned tumor spheroid and tumor tissues
Tumor    Finetek, Tokyo, Japan). Then the tissues were cut into 6 μm thick sections using a cryostat (Leica CM1950, Leica Biosystems, Wetzlar, Germany). DAPI (Roche, Indianapolis, IN, USA) was stained to visualize nuclei of cells for 5 min. The mounted slides allowed fluorescent images to be taken using a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). S18
Tumor volumes were measured and recorded weekly using a caliper. Tumor volumes were calculated using the expression, tumor volume (mm 3 ) = 1/2(length x width 2 ). At the end of the experiment, mice were terminated with CO2 gas and the tumor weights and body weights were measured. No data were excluded from the analyses.

Statistical analysis
For all quantitative data, where statistical analysis was performed, statistically meaningful sample sizes were chosen to allow significant differences between negative and positive controls/treatments with or without irradiation to be observed. All in vitro studies were          Where present, different letters signify datasets that are statistically distinct (p < 0.05).