Preparation and characterization of N-TiO2 samples
The details of preparation of N-TiO2 nanoparticles were described in our previous paper . Briefly, The anatase TiO2 nanoparticles (particle size <25 nm; Sigma-Aldrich, St. Louis, MO, USA) were calcined at a flow rate of 3.5 L/min in ammonia atmosphere at 550°C for 20 min to produce the N-TiO2 nanoparticles. The crystalline phases of the N-TiO2 nanoparticles were determined by Raman spectra to be anatase. The ultraviolet-visible (UV/Vis) diffuse reflectance absorption spectra (Additional file 1: Figure S1) of the N-TiO2 and TiO2 samples were measured with a Jasco V550 UV/Vis spectrophotometer (Jasco, Inc., Tokyo, Japan).
Pure and N-doped TiO2 nanoparticles were autoclaved and dispersed in DMEM-H medium at a concentration of 100 μg/ml, respectively. The samples were ultrasonicated for 15 min before using.
Cell culture and PDT treatment
The human cervical carcinoma cells (HeLa) procured from the Cell Bank of Shanghai Science Academy were grown in Petri dishes in DMEM-H solution supplemented with 10% fetal calf serum in a fully humidified incubator at 37°C with 5% CO2 for 24 h.
The cells were incubated with 100 μg/ml pure or N-doped TiO2 under light-free conditions for 2 h and were then illuminated with a visible light filtered by a bandpass filter (400 to 440 nm) from a Xe lamp (100-W; Olympus, Center Valley, PA, USA) at a power density of 40 mW/cm2 for 5 min. The transmission spectrum of that bandpass filter was shown in Additional file 2: Figure S2. As shown in the figure, the filter could transmit some light with the wavelength below 400 nm. Therefore, the pure TiO2 could still absorb a small amount of the transmitted light.
Measurement of ROS induced by TiO2 or N-TiO2 in aqueous suspensions
For the measurement of photo-induced ROS in TiO2 or N-TiO2 aqueous suspensions, 2′,7′-dichlorfluorescein (DCFH), was used as a probe. The DCFH was converted from the diacetate form DCFH (DCFH-DA) (Sigma-Aldrich) by adding 0.5 ml of 1 mM DCFH-DA in methanol into 2 ml of 0.01 N NaOH and keeping the mixture at room temperature in the dark for 30 min. It was then neutralized with 10 ml sodium phosphate buffer (pH = 7.2) . Pure or N-doped TiO2 in phosphate buffered saline (PBS, 100 μg/ml) were mixed with DCFH (25 μM) before visible light irradiation. The non-fluorescent DCFH can rapidly react with ROS to form fluorescent 2′,7′-dichlorofluorescein (DCF). By measuring the fluorescent intensity, the production of ROS could be estimated.
To measure the generations of specific ROS, two probes were used respectively. Dihydrorhodamine 123 (DHR) is mainly sensitive to O2 ·− and H2O2, and 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]-benzoic acid (APF) is selectively sensitive to OH · . It was already demonstrated that the reactive species H2O2, O2 ·−, and 1O2 did not cause any modification in the fluorescence of the probe APF . Pure or N-doped TiO2 in PBS (100 μg/ml) were mixed with DHR (25 μM, Sigma-Aldrich) or APF (10 μM, Cayman Chemical, Ann Arbor, MI, USA) before irradiation. Upon oxidation, the non-fluorescent DHR or APF is converted to the highly fluorescent Rhodamine 123 or fluorescein.
After the samples were irradiated by a visible light (400 to 440 nm) with a power density of 40 mW/cm2 for different times ranging from 1 to 5 min, the fluorescence spectra were recorded by a spectrometer (F-2500, Hitachi, Brisbane, CA, USA) and the fluorescent intensities were compared.
Rhodamine 123 [2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester] (Beyotime, Jiangsu, China), which could bind specifically to the mitochondria, was used to estimate the MMP. When MMP is decreased, the dye could be released from the mitochondria and the fluorescence vanished. The PDT-treated cells were incubated with Rhodamine 123 (5 μg/ml) for 30 min in the dark at 37°C and then were washed with Dulbecco’s PBS (D-PBS) for three times before the visible light illumination.
Measurement of Ca2+ concentration
To study the intracellular calcium concentration, HeLa cells were loaded with 10 μM Fluo-3 AM (Beyotime) for 30 min at 37°C and followed by washing with D-PBS for three times. Then the cells were incubated for another 20 min to ensure complete cleavage of Fluo-3 AM by the intracellular ester enzyme that releases Fluo-3 before the illumination.
Measurement of intracellular NO
The intracellular NO level was detected using a NO-sensitive fluorescence probe DAF-FM DA [3-amino, 4-aminomethyl-2′,7′-difluorescein, diacetate] (Beyotime). The cells were loaded with 10 μM DAF-FM DA at 37°C in the kit buffer for 20 min and were then gently washed with D-PBS for three times and incubated for another 20 min to ensure that the intracellular DAF-FM DA was completely catalyzed to form DAF-FM by ester enzyme before the illumination.
Cell morphology and cytoskeleton observation
The HeLa cells were fixed with 4% paraformaldehyde for 15 min at room temperature with different time intervals after the illumination. Then they were permeabilized with 0.025% Triton X-100 in D-PBS (Sigma-Aldrich Corp., St. Louis, MO, USA) for 2 min. After washing with D-PBS three times, the cells were treated with 1% bovine serum albumin (BSA) for 2 h at 4°C. The fixed cells were stained with 5 nM Alexa Fluor® 488 phalloidin conjugate (Invitrogen, Eugene, OR, USA) for F-actin labeling for 40 min at 37°C. Meanwhile, 1% BSA was added to the staining solution to reduce nonspecific background staining. The cells were washed with 0.05% PBS-Tween20 three times before microscopic observation.
Microscopy and image analysis
The fluorescence images of cells were observed by a laser scanning confocal microscope (FV-300, IX71; Olympus, Tokyo, Japan) using a 488-nm continuous wave Ar+ laser (Melles Griot, Carlsbad, CA, USA) as the excitation source and a × 60 water objective to focus the laser beam. A 505- to 550-nm bandpass filter was used for the fluorescence images. Each experiment was repeated three times independently.
The fluorescence intensities of MMP, Ca2+, and NO probes from the microscopic images were analyzed with the Olympus Fluoview software. The data were expressed in terms of the relative fluorescence intensity of the probes and expressed as mean ± SD. The fluorescence intensity was averaged from 100 to 150 cells for each experiment.