The synthetic process of DTP was obtained in detail according to the patent specifications (CN 101805362) (17). Initially, meso-5,10,15,20-tetraphenyl-porphyrin was synthesized in propionic acid solvent (Meryer Chemical Technology Co., Ltd., Shanghai, China), using the classical Adler method (18). Meso-5,10,15,20-tetraphenyl-porphyrin (0.01 mol) was added and stirred into 20 ml dichloromethane (Aladdin Industrial Inc., Shanghai, China) at −10°C until dissolved. Subsequently, 65% concentrated nitric acid (1 ml; Aladdin Industrial Inc.) was added to the solution. The nitrated product was reduced with sodium nitrite (Aladdin Industrial Inc.) in dichloromethane at room temperature, until a reduced mixture was gained. The meso-5-(ρ-aminophenyl)-10,15,20-triphenyl-porphyrin was obtained as the main product following separation of the mixture by silica gel chromatography. Next, 0.2 g meso-5-(ρ-aminophenyl)-10,15,20-triphenyl-porphyrin, 0.446 g diethylenetriaminepentaacetic acid dianhydride, 0.046 g dimethylaminopyridine (Aladdin Industrial Inc.) and 0.040 g triethylamine were dissolved in 10 ml dimethyl sulfoxide (DMSO; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in a 25 ml flask. Following agitation for 12 h at room temperature, 5 ml H₂O was added to the reaction solution and a precipitate was formed. The final DTP product was then obtained by crystallizing the precipitate of the mixed solution of DMSO and H₂O. The chemical structure of DTP is shown in Fig. 1. DTP was dissolved in Roswell Park Memorial Institute (RPMI)-1640 medium and stored at 12.5×10³ µM at 4°C, protected from visible light.

The PDT effect of DTP was studied using the HGC27 and SNU-1 human gastric cancer cell lines, which were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The two types of cell were cultured in RPMI-1640 culture medium (Beijing Solarbio Science & Technology Co., Ltd.) with 10% Gibco fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a fully humidified atmosphere with 5% CO₂.

DTP solution was prepared in phosphate-buffered saline (PBS; Beijing Solarbio Science & Technology Co., Ltd.) to produce a solution of 3.12 µM concentration. Subsequently, 100 µl DTP solution was added into a 96-well plate. The ultraviolet (UV)-visible absorption spectrum was recorded on a microplate spectrophotometer (Thermo 3001; Thermo Fisher Scientific, Waltham, MA, USA).

HGC27 or SNU-1 cells were loaded into 96-well plates (1×10⁴/well) and incubated in a humidified incubator containing 95% air and 5% CO₂ until cell attachment to the substratum reached ~80% confluence. Subsequently, 100 µl of various concentrations of DTP were added to the wells and incubated for 24 h. Following incubation, the supernatant was replaced with fresh culture medium supplemented with 10% FBS and the cells were irradiated with 6 or 12 J/cm² of laser light at a wavelength of 650 nm, followed by incubation for an additional 3 h at 37°C. The experiment was divided into groups, identical for HGC27 and SNU-1 cells, as follows: Control groups, including untreated cells, cells treated with PS alone and cells exposed to laser light alone; and treatment groups, including cells treated with DTP of various concentrations in combination with laser light energy. The treatment groups comprised: Group 1, 0.78 µM DTP + 6/12 J/cm²; group 2, 1.56 µM DTP + 6/12 J/cm²; group 3, 3.125 µM DTP + 6/12 J/cm²; group 4, 6.25 µM DTP + 6/12 J/cm² and group 5 12.5 µM DTP + 6/12 J/cm².

Cell viability was assessed using the MTT Cell Proliferation and Cytotoxicity Assay Kit’ (Amresco, Solon, OH, USA). Following photosensitization, 100 µg MTT in 20 µl PBS was added into each well (96-well plates) and the cells (1×10⁴/well) were then incubated for a further 3 h. The reaction was stopped by the addition of 180 µl DMSO. The optical density (OD) of each sample was subsequently measured at a wavelength of 490 nm using a microplate spectrophotometer (Thermo 3001; Thermo Fisher Scientific). Cell survival rate (%) = (OD_treated/OD_control) × 100%.

Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Beyotime Institute of Biotechnology, Haimen, China) were used to detect cell apoptosis induced by DTP-PDT. HGC27 and SNU-1 cells were seeded in 6-well plates, followed by incubation with 6.25 µM DTP for 24 h. The cells were subsequently irradiated at 650 nm with 12 J/cm² laser light. Three hours later, the irradiated cells were collected, rinsed with PBS and stained with Annexin V-FITC (200 µg/ml) for 10 min and 30 µg/ml PI in the dark at room temperature, successively. Immediately, the fluorescence was analyzed in 10,000 cells/sample using a FACScan flow cytometer (FCM; Beckman Coulter, Fullerton, CA, USA). The results were expressed as the percentage of cells exhibiting apoptosis relative to the total number of cells analyzed. Blank control and treatment with DTP or 6 J/cm² laser light alone were included as control groups.

HGC27 and SNU-1 cells were seeded in 24-well plates. Following attachment to the substratum to ~80% confluence, 6.25 µM DTP was added into each well for 24 h, followed by irradiation by a laser at 650 nm with 12 J/cm². The cells were subsequently stained with Hoechst 33342 (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature, washed twice with PBS and exposed to UV illumination using a fluorescent microscope (Leica DMIRE 2; Leica Microsystems GmbH, Wetzlar, Germany) to detect the differences in chromatin condensation and fragmentation between the control and treatment groups. The staining was evaluated by three investigators.

HGC27 or SNU-1 cells were cultured on coverslips (Citoglas; Citotest Labware Manufacturing Co., Ltd., Jiangsu, China) placed in Petri dishes (Citoglas; Citotest Labware Manufacturing Co., Ltd.) with RPMI-1640 culture medium. Once the cells reached 85% confluence, 6.25 µM DTP in serum-free cell culture medium was added to the dishes and cells were incubated for 6 h, followed by incubation with: i) 150 nM MitoTracker Green FM for an additional 30 min or ii) 1.5 µM LysoSensor™ Green DND-189 (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at culture temperature. Following incubation, the staining solution was replaced with fresh culture media and the cells were observed by CLSM (TCS SP8; Leica Heidelberg GmbH, Heidelberg, Germany). The excitation wavelength used for DTP was 405 nm, and the fluorescence excitation and emission wavelengths for MitoTracker Green FM or LysoSensor Green DND-189 were 488 and 520 nm, respectively.

Statistical analysis was performed based on one way analysis of variance. All statistical analyses were performed using SPSS 11.0 software (SPSS, Inc., Chicago, IL, USA). The results were recorded as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference and all experiments were conducted at least 3 times.

Initially, a spectral analysis of DTP was conducted (Fig. 2). The absorption spectrum indicated that DTP had significant absorption peaks at wavelengths of 432, 522, 560, 596 and 652 nm, respectively. Since the absorption was strongest at 432 nm, absorption at 405 nm was evaluated, as this was the closest wavelength which was able to be selected as the excitation laser channel during CLSM analysis. Furthermore, considering the penetration depth of light in PDT proportional to the wavelength, a long wavelength proximal to the red region of the spectrum should be used for laser illumination during treatment with PDT, and 650 nm is a suitable wavelength for the MTT and flow cytometry assays (19).

The photodynamic effect of DTP against human HGC27 and SNU-1 cells was evaluated by MTT assay. Table IA and B list the OD values of each group and Figs. 3 and 4 demonstrate the corresponding cell survival rates following PDT for HGC27 and SNU-1 cells, respectively.

Table IA and Fig. 3 exhibit the MTT assay results for DTP-PDT on HGC27 cells. The cell survival rate following DTP-PDT declined with increasing PS concentration and light dose (Fig. 3). Statistically significant differences were observed between treatment groups 3–5 and the cell control group (P<0.01) following exposure to 6 J/cm² illumination, while following 12 J/cm² treatment, a significant difference was identified between treatment groups 2–5 and the cell control group (P<0.01) (Table IA). Fig. 3 also demonstrated that 0.78 µM PS-mediated PDT only induced slight HGC27 cell death, with ~7.75 and 14.46% cell death at 6 and 12 J/cm² illumination, respectively. However, treatment with 3.12 µM PS was able to induce 46.65 and 54.82% cell death. The highest concentration of DTP (12.5 µM) was found to be highly effective, inducing 70.99 and 74.14% mortality at light doses of 6 and 12 J/cm², respectively. The IC₅₀ of DTP was 4.75 and 2.98 µM at light doses of 6 and 12 J/cm², respectively. DTP alone exerted no marked cytotoxicity on HGC27 cells at any concentration. In addition, laser light alone also did not exert any effects on the growth of HGC27 cells.

SNU-1 cells demonstrated similar MTT results to those observed in HGC27 cells (Table IB; Fig. 4). DTP exerted significant apoptosis induction in SNU-1 cells. A significant difference was observed between treatment groups 3–5 and the cell control group (P<0.01) for light doses of 6 and 12 J/cm², while no difference was detected between treatment groups 1 and 2 and the cell control (P>0.05) (Table IB). The death of SNU-1 cells induced by DTP-PDT was proportional to PS concentration and light dose (Fig. 4). The lower concentrations of 0.78 and 1.56 µM resulted in the death of a small percentage cells at light doses of 6 and 12 J/cm², respectively; however, 12.5 µM DTP induced 65.33 and 67.76% mortality and an IC₅₀ of 4.75 and 6.38 µM, respectively. The data also revealed that DTP or laser treatment alone were nontoxic to SNU-1 cells.

DTP-PDT induced significant cell apoptosis in HGC27 and SNU-1 cells, as determined by FCM analysis (Fig. 5). The apoptotic rates of HGC27 and SNU-1 cells were 61.5±6.56 and 45.9±4.62%, respectively. These values were significantly higher than those of the control groups (P<0.01), which demonstrated 3.1±1.25 and 2.9±0.85% apoptosis in HGC27 and SNU-1 cells, respectively. In the DTP alone group, only 2.5±0.98 and 4.3±1.25% cell apoptosis was detected, while 3.7±1.64 and 1.8±0.75% cell apoptosis were detected in the laser light alone group for HGC27 and SNU-1 cells, respectively. Therefore, no significant differences were detected, in comparison with the cell control (P>0.05).

Morphological changes in the cellular nuclei of each group were observed using fluorescence microscopy (Fig. 6). A large quantity of apoptotic cells were detected following PS-PDT, demonstrating significant characteristics of apoptosis, including chromatin condensation, nucleus fragmentation and apoptotic bodies in HGC27 and SNU-1 cells. By contrast, in the control cell, DTP-alone or light-alone groups, the majority of the cells demonstrated intact, rounded/elliptical nuclei and demonstrated no morphological changes in either of the cell types.

The distribution of DTP within HGC27 and SNU-1 cells was evaluated via fluorescent observation by CLSM (Fig. 7). Following the incubation of cells with DTP and MitoTracker Green Probe or LysoSensor Green Probe, the PS emitted red fluorescence that was excited by the 405 nm wavelength and the MitoTracker Green Probe and LysoSensor Green Probe emitted green fluorescence that was excited by the 488 nm wavelength in the same view. The lysosomal overlay images for HGC27 and SNU-1 cells revealed a large quantity of yellow-green fluorescent spots (Fig. 7, lower panels), indicating co-localization of DTP and the lysosomal probe. Conversely, no overlap was detectable between DTP and the mitochondrial probe for the two types of cell (Fig. 7, upper panels). The distribution of DTP in HGC27 and SNU-1 cells was analogous.