TPP ((4-Carboxybutyl)triphenyl phosphonium bromide), NaN3, N-(3-dimethylamino propyl-N′-ethylcarbodiimide) hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). FBS, penicillin–streptomycin, and trypsin were purchased from Gibco (Grand Island, NY, USA). The ROS Kit (containing DCFH-DA), singlet oxygen sensor green (SOSG), MitoTracker, MitoSOX Red, calcein AM, Propidium iodide (PI), and the J-aggregate-forming lipophilic cation (JC-1) were purchased from Molecular Probes (Eugene, OR, USA). The ATP Determination kit (A22066) and cell lysis buffer (16189) were purchased from Invitrogen (Carlsbad, CA, USA). DMEM medium was purchased from HyClone (Logan, UT, USA). Matrigel and the Annexin V-FITC Apoptosis Detection Kit were purchased from BD Biosciences (San Diego, CA, USA). ICG and the Cell Counting Kit-8 (CCK-8) were purchased from Dojindo (Kumamoto, Japan). Dialysis ultrafiltration tubes and bags with a 10-kDa molecular weight cut-off were purchased from Millipore, Inc. (Billerica, MA, USA). All chemicals and reagents were of analytical grade.
Cells, animals, and tumor xenografts
The human OS cell line MG63 and the doxorubicin-resistant OS cell line (MG63/Dox) were purchased from the American Type Culture Collection (USA). Both cell lines were grown as monolayers in DMEM medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C in a 5% CO2 humidified atmosphere.
Athymic male nude mice (aged 6 weeks, weighing 18–22 g) were obtained from the Laboratory Animal Center of the Chongqing General Hospital (Chongqing, China) and were housed in individual vented cages under specific pathogen-free conditions with a 12 h day/12 h night cycle; food and water were provided ad libitum. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Chongqing General Hospital. MG63/Dox cells (approximately 1 × 107) were resuspended in 100 μl Matrigel and subcutaneously implanted into the right flanks of different mice. Tumor-bearing mice were used for in vivo imaging and for phototherapy when the tumor volume reached 60 mm3.
Synthesis of NGO-PEG-BPEI (PPG), TPP-PPG and TPP-PPG@ICG
NGO was prepared according to the modified-Hummer’s method, starting with the oxidization of graphite sheets, followed by ultrasonication . NGO-PEG (PG) and the NGO-PEG-BPEI (PPG) were prepared according to our previously described method [28-31]. To conjugate TPP with PPG, 5 mg of TPP was dissolved in 4 mL water and activated using EDC·HCl (15 mg) and NHS (15 mg) for 15 min at room temperature. Subsequently, 4 mL of PPG (1.0 mg/mL) solution was added to the reaction mixture and magnetically stirred at room temperature for 24 h. Finally, excess TPP was removed via filtration through a 10-KDa filter (Millipore, Inc.) and washed repeatedly with double-distilled water to obtain PPG-TPP (NGO-PEG equivalent, 0.5 mg/mL).
To synthesize TPP-PPG@ICG, ICG (7.74 mg) was dissolved in 1 mL of anhydrous dimethyl sulfoxide as a stock solution (10 mM) for further use. Two hundred microliters of ICG (10 mM) and 1.8 mL of TPP-PPG (0.5 mg/mL) were mixed and stirred for 24 h at room temperature. Then, the whole system was dialyzed against distilled water for 24 h (molecular weight cut-off: 10 kDa). The final product (TPP-PPG@ICG) was freeze-dried and stored below 4°C for further use.
Fourier transform infrared spectroscopy (FT-IR) spectra were obtained for TPP-PPG@ICG using a Nicolet 6700 spectrometer (Thermo Scientific) to confirm that ICG was loaded onto the NGO. An atomic force microscope (AFM) instrument (Bruker Dimension Icon) was used to characterize the size and thickness of TPP-PPG@ICG. The optical properties of TPP-PPG, ICG, and TPP-PPG@ICG were characterized using a Ultraviolet–visible–infrared (UV–vis–NIR) spectrometer (UV-3600 Scanning Spectrophotometer, Shimadzu, Japan). Nanoparticle stability was tested as follows: TPP-PPG or TPP-PPG@ICG was incubated with PBS, DMEM medium, or serum, and the mixtures were monitored for the appearance of precipitates. The release of ICG from TPP-PPG@ICG was studied by adding the material to acidic (pH = 5.0) or alkalescent (pH = 7.4) PBS at 37℃ or 43℃, respectively. To determine the release kinetics of ICG from TPP-PPG@ICG, PBS was collected after centrifugation and replaced with the same volume of PBS at each sampling time. The amounts of ICG released from TPP-PPG were evaluated using a UV–vis–NIR spectrometer for loading-efficiency measurements. To determine the ICG loading on TPP-PPG, TPP-PPG@ICG solution was diluted in 5 mL ethyl acetate/ethanol (9:1, v/v) and sonicated for 30 min to completely release ICG. ICG levels were determined by measuring the UV–vis-NIR absorption spectra. ICG loading was defined as follows: ICG content (%, w/w) = (ICG weight in TPP-PPG@ICG/TPP-PPG weight) × 100%. All the measurements were performed in triplicate.
Single oxygen detection
The generation of singlet oxygen (1O2) was evaluated using SOSG. Typically, solutions containing ICG, TPP-PPG, and TPP-PPG@ICG (ICG concentration = 10 μM) were mixed with SOSG, which was dissolved in water containing 2% methanol to a final concentration of 1 mM. Then, the mixture was immediately irradiated with a laser at 808 nm for 5 min (energy density: 0.6 W/cm2). The emission peak of SOSG at 530 nm was obtained by excitation with a light source at 494 nm, and the data were quantified for singlet oxygen generation.
Measurements of photothermal performance
One milliliter each of PBS, ICG, TPP-PPG, and TPP-PPG@ICG in aqueous solution was placed in a quartz cell and irradiated at 808 nm with a power density of 0.6 W/cm2 for 10 min at pre-designed time intervals (0 min, 2.5 min, 5.0 min, 7.5 min, and 10.0 min). The temperature was measured using an infrared thermometer.
Cellular uptake and intracellular localization
MG63/Dox cells were seeded in 6-well plates at a density of 1 × 105 cells/well. After overnight incubation, TPP-PPG@ICG was added at a final concentration of 10 μM. After 1 h, 4 h, 8 h, 12 h, or 24 h of incubation, the cells were washed thrice with PBS, trypsinized, resuspended in medium, and harvested for analysis using flow cytometry using a FACSVerse instrument (BD Biosciences). The mean FL intensity of 1 × 104 cells was recorded for each sample.
To study subcellular localization, MG63/Dox cells (1 × 105 cells per mL) were exposed to TPP-PPG@ICG (ICG concentration = 10 μM) on 35-mm glass-bottom dishes for 24 h. After rinsing thrice with PBS, the cells were treated with MitoTracker for 10 min to stain the mitochondria. Then, the cells were washed thrice with PBS before capturing images using a confocal laser-scanning microscope (CLSM).
In vitro analysis
MG63/Dox cells were seeded in 96-well plates (5 × 103 cells/well in 100 µl) and incubated for 24 h. Then, ICG, TPP-PPG, PPG@ICG, or TPP-PPG@ICG was added to various concentrations of ICG. Subsequently, the cells in the non-irradiated groups were rinsed with PBS and incubated for another 24 h without laser irradiation. The cells in the irradiated groups were exposed to 808-nm laser light at 0.6 W/cm2 for 5 min and incubated for another 24 h. Then, cell viabilities were assessed by performing CCK-8 assays. In addition, cell viabilities were also assessed in the presence of NaN3 or at low temperature to verify the participation and individual therapeutic efficacies of PDT and PTT. MG63/Dox cells were seeded in 96-well plates (5 × 103 cells/well in 100 µl) and incubated for 24 h. Then, TPP-PPG@ICG was added at various concentrations. NaN3 was added to cell culture medium at 100 mM to quench singlet oxygen molecules and thereby block the effect of PDT. Cells were irradiated at 4°C to maintain a constant temperature and nullify the effect of PTT. Negative-control groups were treated with the laser only or with TPP-PPG@ICG without irradiation. The cells in the irradiated groups were exposed to 808-nm laser light at 0.6 W/cm2 for 5 min and incubated for another 24 h. Subsequently, cell viabilities were assessed by performing CCK-8 assays.
Calcein AM/PI co-staining was also performed to assess the synergistic phototherapeutic effect of TPP-PPG@ICG. For visualization, MG63/Dox cells (1 × 105 cells per well) were first seeded in 6-well plates and incubated overnight. Then, the cells were treated with equivalent dosages of PBS (-), TPP-PPG@ICG (-), ICG (+), TPP-PPG (+), PPG@ICG (+), or TPP-PPG@ICG (+) (NGO 20 μg/mL; ICG 15 μM) for 24 h, where “(-)” indicates that no irradiation was applied, and “(+)” indicates that the cells were irradiated at 0.6 W/cm2 for 5 min. After incubation for another 24 h, the cells were incubated with calcein AM (to visualize live cells) and PI (to visualize dead/late apoptotic cells), according to the manufacturer’s suggested protocol.
Detection of apoptosis and intracellular ROS
MG63/Dox cells were seeded overnight in 6-well plates (2 × 105 cells per well) and then treated for 24 h with equivalent dosages of PBS (-), TPP-PPG@ICG (-), ICG (+), TPP-PPG (+), PPG@ICG (+), or TPP-PPG@ICG (+), where “(-)” indicates that no irradiation was applied, and “(+)” indicates that the cells were irradiated at 0.6 W/cm2 for 5 min) (NGO equivalent, 20 μg/mL; ICG equivalent, 15 μM). The cells were collected at 6 h post-laser irradiation after careful trypsinization and low-speed centrifugation, followed by washing twice with PBS. The collected cells were resuspended in 100 μL binding buffer and stained with 2 μL annexin V-FITC and 2 μL PI for 15 min at room temperature in the dark. After staining, the cells were collected via low-speed centrifugation, washed twice with PBS, and diluted with 400 μL binding buffer for flow cytometric analysis using an Epics XL-MCL instrument (Beckman Coulter).
The 2′,7′-dichlorofluoreseindiacetate (DCFH-DA) Kit was used to detect the intracellular ROS generation. MG63/Dox cells were seeded overnight in 24-well plates (1 × 105 cells per well) and then treated with equivalent dosages of PBS (-), TPP-PPG@ICG (-), ICG (+), TPP-PPG (+), PPG@ICG (+), or TPP-PPG@ICG (+) (NGO equivalent, 20 μg/mL; ICG equivalent, 15 μM) for 24 h, where “(-)” indicates that no irradiation was applied and “(+)” indicates that the cells were irradiated at 0.6 W/cm2 for 5 min. After irradiation, the cells were promptly washed with PBS and incubated with 10 μM DCFH-DA at 37°C for 30 min. ROS FL signals were evaluated using a DMLRB inverted FL microscope (Leica).
Detecting the mitochondrial membrane potential
Changes in the mitochondrial membrane potential were measured using JC-1 and imaged using a CLSM. Briefly, MG63/Dox cells (1 × 105 cells per well) were incubated with equivalent dosages of PBS (-), TPP-PPG@ICG (-), ICG (+), TPP-PPG (+), PPG@ICG (+), or TPP-PPG@ICG (+) (NGO equivalent, 20 μg/mL; ICG equivalent, 15 μM), where “(-)” indicates that no irradiation was applied and “(+)” indicates cases where the cells were irradiated for 24 h. The irradiated groups were then illuminated using a laser at 808 nm (0.6 W/cm2, 5 min) and incubated for another 24 h. The non-irradiated groups were incubated for 48 h under the same conditions. Then, the cells were promptly washed with PBS and incubated with 5 mM JC-1 at 37°C for 30 min. The cells were rinsed again with PBS and analyzed using a CLSM.
Detection mitochondrial superoxide levels
Mitochondrial superoxide generation was assessed by measuring MitoSOX FL using a CLSM. Briefly, MG63/Dox cells were seeded in glass-bottom 35-mm plates overnight and then treated with equivalent dosages of PBS (-), TPP-PPG@ICG (-), ICG (+), TPP-PPG (+), PPG@ICG (+), or TPP-PPG@ICG (+) (NGO equivalent, 20 μg/mL; ICG equivalent, 15 μM), where “(-)” indicates that no irradiation was applied and “(+)” indicates cases where the cells were irradiated at 0.6 W/cm2 for 5 min. After treatment, the cells were washed twice with PBS, incubated with 5 µM MitoSOX for 10 min, washed twice with PBS, and analyzed using a CLSM.
ATP determination assay
A standard curve was generated for a series of ATP concentrations, using an ATP Determination Kit. MG63/Dox cells were seeded in 96-well plates (1 × 104 cells per well) in 100 μL of DMEM and incubated for 24 h prior to performing ATP-determination assays. Then, the cells were treated with equivalent dosages of PBS (-), TPP-PPG@ICG (-), ICG (+), TPP-PPG (+), PPG@ICG (+), or TPP-PPG@ICG (+) (NGO equivalent, 20 μg/mL; ICG equivalent, 15 μM), where “(-)” indicates that no irradiation was applied and “(+)” indicates cases where the cells were irradiated at 0.6 W/cm2 for 5 min. After 6 h of incubation, the culture medium was removed, and the cells were treated with lysis buffer. Next, the reagents of the ATP Determination Kit were added to the lysed cells, and a plate reader was used to measure the luminescence in order to calculate each ATP concentration. Each experiment was repeated thrice, and mean values were calculated.
NIR FL and thermal imaging
In vitro thermal imaging of the PBS blank, ICG, and TPP-PPG@ICG under irradiation (808 nm, 0.6 W/cm2, 5 or 10 min) was conducted using an infrared thermal-imaging camera (Ti32, Fluke, USA). TPP-PPG@ICG was injected via the tail vein (0.5 mg/kg) into athymic nude mice bearing MG63/Dox tumor xenografts to evaluate the tumor-targeting ability, NIR FL, and thermal imaging. In vivo NIR FL imaging was performed using an NIR imaging system (Kodak). All the sets and imaging conditions were the same as those of the reported method . The major organs that were excised were imaged after sacrificing the nude mice that were intravenously administered with TPP-PPG@ICG at 24 h post-injection. Regional body temperatures and infrared thermographic maps were obtained using a Ti27 infrared thermal imaging camera (Fluke).
Combined in vivo treatment with PDT and PTT
When the tumor volume reached 60 mm3, the tumor-bearing mice were randomly divided into six groups, with five mice per group. These groups included the PBS-treated, non-irradiated group (control group); the TPP-PPG@ICG-treated, non-irradiated group; the ICG-treated, irradiated group; the TPP-PPG-treated, irradiated group; the PPG@ICG-treated, irradiated group; and the TPP-PPG@ICG-treated, irradiated group. Equivalent dosages of the therapeutic agents were injected intravenously (ICG equivalent, 750 μM). In addition, for the irradiation groups, laser irradiation at 808 nm (0.6 W/cm2, 5 min) was applied at the tumor site. Variations in tumor volumes and body weights in each group were monitored every three days for up to 15 days to evaluate the therapeutic effects. Then, the animals were euthanized on day 15. The tumors and major organs (the heart, liver, spleen, lungs, and kidneys) were collected. H & E and TUNEL staining of tumor sections from different groups were performed, and the stained samples were observed under a bright-field microscope (Olympus)
In vivo toxicity assessment
The 200μL TPP-PPG@ICG (ICG equivalent, 750 μM) was injected intravenously into five healthy male nude mice. Another five nude mice injected with normal saline were selected as the control group. Then, at days 30, the blood was collected from each mouse for the blood chemistry test and complete blood panel analysis.
All statistical analyses were performed with SPSS 13.0 software. Data were presented as mean ± standard deviation. The significance of the data is analyzed according to a Student’s t-test: *P < 0.01.