Dulbecco’s modified eagle medium (DMEM) high glucose, phosphate-buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsin/EDTA, fetal bovine serum (FBS)-gamma irradiated, antibiotic antimycotic solution-100X (10,000 U penicillin, 10 mg streptomycin, and 25 µg amphotericin B per ml in 0.9% normal saline) were obtained from Himedia, Mumbai, India. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE), prodan, superoxide dismutase (SOD) from bovine erythrocytes (≥ 3000 U/mg protein), superoxide dismutase -polyethylene glycol (SOD-PEG) from bovine erythrocytes (≥ 1350 U/mg protein), propidium iodide (PI), and β-NADPH were obtained from Sigma, USA. Singlet oxygen sensor green (SOSG) and 3′-(p-aminophenyl) fluorescein (APF) were obtained from Thermo Fischer Scientific, USA.

2.1 Cell culture

Human breast carcinoma (MCF-7) and human cervical adenocarcinoma (Hela) cells were obtained from NCCS, Pune, India. Cells were maintained in DMEM supplemented with 10% FBS and 1X antibiotic antimycotic solution under 5% CO2 and 95% air atmosphere in a humidified incubator (ESCO, Singapore) at 37 °C. The cells after ~ 80% confluent growth were trypsinized, re-suspended in growth medium, and plated at required cell density in flat-bottomed 96-well plates or 3.5 cm Petri dishes. The cells were allowed to grow for 16–18 h and then used for the experiments.

2.2 Photosensitizer preparation

Cycloimide Purpurin-18 (CIPp-18) was prepared from Purpurin-18 following the procedure described by Mironov et al. [12]. Briefly, ethanolic solution of Purpurin-18 was treated with hydrazine hydrate (N2H4·H2O) for 48 h in dark at room temperature followed by acidification by 1N HCl for 2 h. The reaction mixture was diluted to twice its initial volume with de-ionized water and was partitioned in dichloromethane (DCM). The organic phase was removed and evaporated to dryness in vacuum. The product was purified on a silica column using methanol:acetonitrile (7:3) solvent system. A concentrated stock solution of CIPp-18 was prepared in methanol:PEG 400 (1:1) and stored in dark under cold conditions.

2.3 Spectroscopy

The absorption spectra of CIPp-18 in different solvents were recorded on Cintra-20 spectrophotometer (GBC, Australia) in the wavelength range from 350 to 750 nm with 1.0 nm bandpass. The fluorescence emission spectrum of CIPp-18 was recorded from 650 to 750 nm on FLS900 fluorescence spectrometer (Edinburgh Instruments, UK) at 416 nm excitation wavelength, ~ 0.5 nm band pass, and ~ 2 nm excitation and emission slit widths. Mass spectrum of methanolic solution of CIPp-18 was recorded on MicroTOF-Q11 mass spectrometer (Bruker, USA). 1H NMR spectrum of CIPp-18 was determined using Ascend™ 500 NMR spectrometer (Bruker, USA) in deuterated chloroform (CDCl3).

2.4 n-Octanol/PBS partition coefficient (log P)

Lipophilicity of CIPp-18 was determined using Octanol/water partition coefficient method. Briefly, a mixture containing equal volume of 1-octanol and PBS (pH 7.4) was vortexed vigorously for 20 min to prepare a solution of octanol saturated with PBS and PBS saturated with octanol. The mixture was allowed to stand and then centrifuged at 12.3 X 103 g for 10 min. The octanol and PBS phases were separated and 50 µM CIPp-18 was added into each phase. After addition of CIPp-18, octanol and PBS were mixed and the mixture was shaken vigorously for further 20 min and centrifuged at 12.3 X 103 g for 10 min. The octanol and PBS phases were separated and ~ 50 µL octanol/PBS phase was diluted in methanol to obtain 5% octanol/PBS in final methanolic solution. The concentration of CIPp-18 in the methanolic solution of each phase was determined spectrophotometrically. Log P value was determined using the following formula:

Log Poct/PBS= Log [Coct/ CPBS],

where Coct and CPBS are absolute concentrations of CIPp-18 in the octanol and PBS phase, respectively. All the measurements were carried out at room temperature.

2.5 Irradiation

Irradiation was performed using Near-Infrared (NIR) light (700 ± 25 nm) emitted from a light source LC-122A (Ci tek, USA). The light source was coupled to an optical fiber probe (Dia. 1.2 cm, length 1 m) with an in-built narrow band pass filter. The distal end of fiber optic probe was set at a height of 11.5 cm to expand the beam area for irradiation of set of 5 columns of 4 wells each in a 96-well plate. Power density at the sample level was ~ 3.0 mW/cm2 and was uniform within the area of illumination as determined using an optical power meter (Newport, CA, USA).

2.6 Determination of ROS generation in photochemical reaction

The capability of CIPp-18 to generate 1O2 was determined in aqueous buffer system using SOSG which reacts selectively with 1O2 to produce a highly fluorescent endoperoxide product. The reaction mixture contained 1% Cremophor EL (CrEL) in 50 mM sodium phosphate buffer (pH ~ 7.4), 500 nM CIPp-18, and 1.0 µM SOSG. Cremophor EL was added to the solution to prevent aggregation of CIPp-18. The reaction mixture was prepared and dispensed in wells of 96-well plate (100 µl/well) in dark. To verify that change in fluorescence of SOSG is due to 1O2, separate reaction mixtures containing 3 mM sodium azide or 40 mM histidine and a reaction mixture prepared in D2O were used. Further, a reaction mixture purged with nitrogen for 30 min was also used to check effect of hypoxia on 1O2 generation. To maintain hypoxia, each well was filled with 300 μl reaction mixture, such that the air column above the well was reduced to minimum, and then, the 96-well plate was immediately sealed within a vacuum zipper bag. The wells in different rows were irradiated with NIR light (700 ± 25 nm, 3.0 mW/cm2) for 2–10 min. A separate row of wells protected from light was used as dark control. For each sample, at least four wells were used as replicates. After irradiation, the fluorescence of the SOSG endoperoxide product was recorded on a multi-mode plate reader (Biotek Synergy H1, USA) keeping excitation and emission wavelength at 485 nm and 530 nm, respectively. The relative yield of singlet oxygen is expressed as F–F0 where F and F0 is the fluorescence intensity of irradiated samples and control (no CIPp-18, no light), respectively.

The generation of O2·– in CIPp-18 induced photochemical reaction was determined by Nitroblue Tetrazolium (NBT) assay which is based on formation of blue colored formazan subsequent to reaction of NBT with O2·–. The reaction mixture contained 5.0 µM CIPp-18, 24.0 µM NBT, 26.0 µM β-NADPH and 1% CrEL in 50 mM sodium phosphate buffer (pH 7.4). The reaction mixture (1.0 mL) was taken in a quartz cuvette (Vol—2.0 mL, path length—1.0 cm) and irradiated with NIR light (700 ± 25 nm, 3.0 mW/cm2) for different time periods. To check effect of hypoxia on O2·– generation, the cuvette was completely filled with deoxygenated reaction mixture and sealed with air tight cap, such that no air column remained at the top. Prior to irradiation and at each time point after irradiation, the absorbance of the reaction mixture at 550 nm was recorded on a Cintra-20 spectrophotometer (GBC, Australia). To confirm that the change in absorbance is due to generation of O2·–, a reaction mixture containing superoxide dismutase (4.0 U/ml) was used. The relative yield of O2·– generation was expressed as A–A0, where A and A0 is absorbance of the irradiated and control (no CIPp-18, no light) samples, respectively.

2.7 Photodynamic treatment of cancer cells

MCF-7 and Hela cells were subcultured and plated in 96-well plates at cell density of 7500 cells/well as described above. After removing the growth medium from each well, fresh growth medium containing 2.0 µM CIPp-18 was added and cells were incubated for 3 h in 5% CO2/95% air atmosphere at 37 °C in dark. Post-incubation, the cells were washed once with fresh growth medium and then irradiated with NIR light (700 ± 25 nm) as described above. The light dose was varied from 0.36–1.40 J/cm2 by varying the irradiation time from 2 to 8 min. Subsequently, cells were incubated for ~ 18 h at 37 °C in dark, and then, cell viability was determined by MTT assay.

2.8 Detection of morphological features of PDT-induced cell death

PDT-induced changes in cellular and nuclear morphology were imaged by staining cells with Hoechst (HO) and Propidium Iodide (PI). HO is a cell permeable DNA-binding dye which gives intense blue fluorescence upon binding with condensed DNA, a hallmark for apoptosis. PI is a cell impermeable red fluorescent dye which enters the cells when membrane integrity is lost due to necrosis [17]. Briefly, cells grown on glass coverslips were subjected to PDT (2.0 µM CIPp-18, 1.4 J/cm2 (LD90)) and then incubated at 37 °C for ~ 18 h. Subsequently the cells were incubated in serum-free medium containing ~ 23 µM HO and 10 µM PI for 10 min. After incubation, the cells were washed twice with serum-free medium (w/o phenol red) and imaged on confocal microscope at 40X magnification. HO was excited using 405 nm laser and fluorescence emission was recorded within 416–520 nm. PI was excited using 560 nm laser and emission from 570 to 680 nm was imaged. Fluorescence of both the cellular stains were recorded using the PMT detector.

2.9 PDT under hypoxic condition

To assess the phototoxicity induced by CIPp-18 under hypoxic conditions, MCF-7 and Hela cells post-incubation with 2.0 µM CIPp-18 were transferred to serum-free medium which was deoxygenated by nitrogen flushing for 30 min. To maintain cells under hypoxic condition, we used the procedure described earlier by Mattheisen et al. [18] with slight modification that instead of vacuum sealing machine, we used a vacuum suction pump. In brief, immediately after adding deoxygenated serum-free media, the 96-well plate was placed in a vacuum zipper bag. The bag was flushed with nitrogen and was immediately vacuum sealed. Consistency of hypoxia for the course of experiment was tested with a dye indicator solution containing Methylene blue, Glucose, and KOH (Supl. Figure 2). The cells were incubated for 1 h at 37 °C in dark and then subjected to PDT at LD50 (0.72 J/cm2) and LD70 (1.2 J/cm2) light dose under hypoxic condition maintained within vacuum zipper bag. Post-PDT, deoxygenated media was replaced with fresh growth media and the cells were incubated for ~ 18 h at 37 °C in dark before performing the cell viability assay.

2.10 Cell viability assay

At ~ 18 h after irradiation, cell viability was measured using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay. Briefly, the growth medium from each well was removed and replaced with serum-free medium containing 0.5 mg/ml MTT. The cells were incubated at 37 °C for 3 h in dark. After incubation, MTT containing medium was removed and 100 µl DMSO was added in each well to dissolve the formazan crystals formed within the cells. The plate was left at room temperature for 15 min to allow complete solubilisation, and then, the absorbance of each sample was recorded at 570 nm using a microplate reader (Power Wave 340, Bio-tek instruments Inc., USA). The percent viability was calculated relative to the absorbance value of the control sample which received no CIPp-18 and light treatment. To check for dark toxicity, cells treated with CIPp-18 but not subjected to irradiation were used as dark control. For control as well as treatment, four wells were used as replicates in each experiment.

2.11 Detection of intracellular ROS generation

Intracellular generation of 1O2 and other ROS subsequent to PDT with CIPp-18 was monitored by confocal microscopy using fluorescent probes DCFH-DA, APF, and DHE which detects total ROS, ·OH/1O2, and O2·–, respectively. In brief, MCF-7 cells plated on glass cover slips at cell density of 5 × 105 cells/mL in a 35 mm glass bottom petri dish were incubated in growth medium containing 2.0 μM CIPp-18 for 3 h at 37 °C, in dark. Cells were washed once with serum-free media followed by addition of fresh serum-free medium (w/o phenol red) containing either DCFH-DA (20 μM), APF (20 μM) or DHE (10 μM). The cells were re-incubated for 90 min to allow uptake of fluorescence probes. Post-incubation, the cells were exposed to NIR light at a dose of 1.2 J/cm2 (LD70). To study intracellular ROS generation under hypoxic condition, the cells were first incubated with fluorescence probes for 30 min in serum-free media, and then, the media was replaced with deoxygenated media containing same fluorescence probe. The petri dishes were sealed in vacuum zipper bags and re-incubated for 1 h at 37 °C, in dark. After incubation, cells maintained under hypoxic conditions in vacuum zipper bags were irradiated with NIR light as mentioned above. The fluorescence of different probes in cells was imaged immediately after irradiation on a confocal microscope (LSM 880, Carl Zeiss, Germany) at 40X magnification (NA 1.4) using 488 nm laser line for excitation and bandwidth of emission channel as 490 nm-600 nm.

2.12 Determination of involvement of ROS in phototoxicity

MCF-7 cells grown in 96-well plates were incubated with 2.0 µM CIPp-18 for 3 h in dark. After incubation, the cells were washed with serum-free media and incubated for 1 h in serum-free media containing either sodium azide ( 3.0 mM), histidine (40 mM), DMSO (0.1%), mannitol (40 mM), cyanocobalamin (2 mM), or serum-free media prepared in deuterium oxide (D2O). After incubation, the cells incubated with ROS quenchers were irradiated with NIR light at LD80 (~ 1.3 J/cm2) light dose. Since D2O was expected to enhance the phototoxicity, the cells incubated with D2O media were irradiated with NIR light at a lower dose (~ 0.5 J/cm2) corresponding to LD30. After PDT, probe containing medium was removed and replaced with fresh growth medium. The cells were incubated at 37 °C in dark for ~ 18 h, and then, cell viability was determined by MTT assay.

2.13 Membrane localization of CIPp-18

Localization of CIPp-18 in membrane was assessed with the help of cell membrane specific fluorescent dye Prodan. MCF-7 cells grown as monolayer on coverslips were incubated with 2.0 µM CIPp-18 in growth medium for 3 h in dark. After incubation, medium was removed and replaced with serum-free medium (w/o phenol red) containing 5.0 µM Prodan for 30 min. Subsequently, cells were washed twice with serum-free medium (w/o phenol red) and imaged under 40X magnification on a confocal microscope. The fluorescence of Prodan excited with 488 nm argon ion laser line was recorded on PMT channel between 517 and 585 nm. Since CIPp-18 at concentration used showed weak fluorescence on PMT detector, its intracellular fluorescence excited with 405 nm diode laser was recorded on a high-sensitivity GaAs detector using appropriate emission filters (LP610 and BP690/50). We also imaged the unstained cells and cells stained with Prodan on GaAsp detector using same settings, and it was verified that autofluorescence of cells or fluorescence of Prodan was not mixed with the fluorescence of CIPp-18.

2.14 PDT-induced changes in cell morphology

Cells grown on glass coverslips were subjected to PDT (2.0 µM CIPp-18, 1.2 J/cm2 light dose) under normoxic and hypoxic conditions as described above. Subsequently, the cells were incubated at 37 °C for ~ 4 h to allow morphological changes to occur and then stained with ~ 10.0 µM PI for 10 min in serum-free medium (w/o phenol red). After washing, the cells twice with serum-free medium (w/o phenol red) their brightfield and fluorescence images were recorded at 40X magnification on a confocal microscope. PI was excited with 560 nm laser and its emission between 570 and 680 nm was imaged on PMT detector.

2.15 Statistical analysis

All the experiments were performed at least three times using four replicates in each experiment. The data obtained from three independent experiments were plotted as mean ± standard deviation. Origin Pro-2018 (USA) software was used to plot graphs and perform statistical analysis. The significance between the means of two groups was determined by a Student’s t test. p < 0.05 was considered to be statistically significant.