IR-780 Dye as a Sonosensitizer for Sonodynamic Therapy of Breast Tumor

Sonodynamic therapy (SDT) has become a new modality for cancer therapy through activating certain chemical sensitizers by ultrasound (US). Discovery and development of novel sonosensitizers are attracting extensive attentions. Here, we introduce IR-780 iodide, a lipophilic heptamethine dye with a peak optical absorption of 780 nm wavelength, which can function as SDT agents for breast cancer treatment. The in vitro cellular uptake, cell viability, and the generation levels of reactive oxygen species (ROS) were examined by using 4T1 breast cancer cells incubated with various concentrations of IR-780 followed by US irradiation. Our results showed a dose- and time-dependent cellular uptake of IR-780 iodide in 4T1 cancer cells. Significant lower viabilities and more necrotic/apoptotic cells were found when these cancer cells were treated with IR-780 iodide with US irradiation. Further analyzing the generation of ROS demonstrated significant increase of 1O2 level and H2O2, but not ⋅OH in the SDT-treated cells. The in vivo anti-tumor efficacy of SDT with IR-780 revealed significant tumor growth inhibition of xenografts of 4T1 cancer cells; it was further confirmed by histological analysis and TUNEL staining. Our results strongly suggest that SDT combined with IR-780 may provide a promising strategy for tumor treatment with minimal side effects.

irradiated by US 14 . To date, more and more sonosensitizers are found and applied to the cancer treatment 15 . Some photosensitizers including porphyrin derivatives and phthalocyanins attract the most extensive attentions as sonosensitizers 16,17 .
IR-780 iodide, a lipophilic cation heptamethine dye (the molecular structure is shown in Fig. 1) with the peak optical absorption at 780 nm wavelength, has emerged as a potential fluorescent probe for in vivo tumor imaging due to its high fluorescence intensity, excellent stability and preferential accumulation in the tumor 18 . In addition, IR-780 can be used for photothermal therapy (PTT) because of its strong absorption at near-infrared wavelengths 19 . Recently, the PDT application of IR780 has also been reported 20 . In this study, we investigated the feasibility of IR-780 as a sonosentizer in the SDT. To our best knowledge, it is the first report about the application of SDT using IR-780 as a sonosensitizer for treatment of breast cancer.
In vitro cellular uptake of IR-780 iodide. IR-780 iodide was dissolved in DMSO and diluted with PBS in final concentration of 100 mM for vitro experiment. For cellular uptake study, 4T1 cells (2 × 10 6 cells/dish) were seeded in 35 mm diameter dishes and cultivated for 24 h for full adhesion. The media were replaced with the fresh media containing 4 μ M, 10 μ M or 16 μ M of IR-780 iodide. The medium containing the same volume PBS were used as the control. After 1, 2 or 3 h, the cells were washed with PBS for 3 times and harvested with trypsin digestion. After that, the cells were detected by flow cytometry (BD FACSCanto II, USA).
To further determine the cellular distribution and retention of IR-780 in 4T1 cells, a laser confocal microscopy (Leica TCS SP5, Wetzlar, Germany) was used. Briefly, 4T1 cells were seeded onto 8-well chambered cover-glass at a density of 2 × 10 4 /well (0.4 mL). 24 h later, the medium was changed with fresh medium containing 4 μ M, 10 μ M or 16 μ M of IR-780 iodide. After 1 h incubation, the cells were washed and fixed with 4% paraformaldehyde solution for 20 min, then stained with Hoechst 33258 for 5 min. The cells were examined under a confocal laser scanning microscope.
In vitro cytotoxicity of IR-780 iodide with US irradiation. The cell cultivation and IR-780 treatment were the same as stated above. After washing cells for 3 times, 2 mL of fresh complete medium were added into each dish. All of groups were exposed to US irradiation using a sonicator device purchased from Tianjin Tianshi Technology Company (Tianjin, China). The in vitro US experiment instrument was showed in Supplementary  Fig. 1. Briefly, the transducer was immerged in a sink filled with degassed water. There was a dish holder on the water surface to fix the cell dish which was not fully immersed in the tank where the transducer is located. The distance between the transducer and the dishes was kept at 3 cm. US irradiation was performed at the spatially and temporally averaged intensity of 1.5 W/cm 2 (frequency: 1 MHz; duty cycle: 50%, pulse repetition frequency: 1 Hz) for 20 s or 40 s, respectively. After US treatment, the cell viability was assessed by CCK-8 detection kits. The absorbance at 450 nm was determined using a multimode plate reader (Synergy ™ 4, BioTek, VT, USA).
For further assess of apoptosis induced by US and IR-780, the 20-sencond-US-treated cells were stained with FITC-conjugated Annexin V and propidium iodide for 15 min and analyzed by flow cytometry (BD Accuri C6, USA).
Detection of ROS levels. The intracellular ROS were detected after the cells were treated with US and IR-780. As for 1 O 2 detection, the probe reagents were dissolved with DMSO at the concentration of 5 mM, and then diluted with PBS at the final concentration of 5 μ M for experiment use. 4T1 breast cancer cells (1 × 10 6 /dish) were seeded in 35 × 35 mm dishes and cultivated for 24 h for full adhesion. The medium was replaced by the fresh medium containing 10 μ M of IR-780 iodide. As a control, no IR-780 was used. After being incubated for 3 h, the cells were washed and added with the prepared probe solution. Then cells were treated with US irradiation at 1.5 W/cm 2 for 20 s. After treatment, the cells were harvested, and the fluorescence intensity was measured using a multimode plate reader at the excitation wavelength of 510 nm and emission wavelength of 580 nm. Relative fluorescence units (RFU) of samples were calculated and normalized to the untreated cells. As for the detections of H 2 O 2 and ⋅ OH, CM-H2DCFDA (final concentration of 7.9 μ M) and HPF (final concentration of 10 μ M) were used, respectively. Similar procedures were performed as stated above. The fluorescence intensity of CM-H2DCFDA was determined at the excitation wavelength of 490 nm and emission wavelength of 525 nm. The fluorescence intensity of HPF was measured at the excitation wavelength of 490 nm and emission wavelength of 515 nm.
Effect analysis of active oxygen scavengers. The effects of active oxygen scavengers were detected according to the previous report 21 . Briefly, the cell cultivation were the same as stated above. 10 μ M of IR-780 iodide was added and incubated for 3 h. After washing out the free IR-780 dye, 2 mL of fresh complete medium were added into each dish. As the control, no IR-780 was used for these cells. Before ultrasound irradiation, histidine (10 mM), mannitol (100 mM) or SOD (100 ug/mL) were added to the cells and US irradiation was performed for 20 s. After that, cell damage induced by insonation in the presence and absence of IR-780 with active oxygen scavengers was detected by staining of the cells with Trypan Blue dye as described previously 1 . In vivo imaging analysis. All animal studies were approved by the Institute's Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. The methods were carried out in accordance with the approved guidelines. 6-to 8-week-old female BALB/c mice (five per cage) were housed on a 12:12 light: dark cycle with free access to food and water. 4T1 breast carcinoma cells (1 × 10 6 /each) were injected into the right flank of mice. When mice had developed palpable tumors 6 days after tumor cell injection (about 5 mm in diameter), 0.1 mL 800 μ g/mL of IR-780 was injected into the tumor. Images and fluorescence semi-quantitative analysis of IR-780 were taken at 0.1 h, 0.5 h, 3 h, 6 h after injection using the ex/in vivo imaging system (CRi maestro, USA) with a 704 nm excitation wavelength and a 740 nm filter to collect the fluorescence signals of IR-780. 48 h later, the mice were sacrificed and tissue organs were collected for imaging and biodistribution analysis.

Anti-tumor efficacy of SDT with IR-780.
In the in vivo experiments, we set up the experiment instrument as shown in Supplementary Fig. 2. Briefly, the transducer was immersed in a degassed water-filled sink. On the surface of water, there is a mouse supporting plate with a hole (15 mm diameter) at the center of the plate. The mouse was placed on the plate and the tumor was positioned into the hole. Tumor-bearing mice were randomly divided into 4 groups (5 animals each group). Mice were intratumorally injected with 100 μ L PBS, 100 μ L PBS + US, 80 μ g of IR-780 (in 100 μ L solution), or 80 μ g of IR-780 + US. US irradiation was performed at 2 W/cm 2 for 4 min. The day when mice were received with US treatment was designated as day 0. The tumor volumes and body weights of the mice were recorded. The tumor volumes were calculated by the following formula: Tumor volume (mm 3 ) = axial length (mm) × (lateral axial length) 2 (mm 2 )/2. Histological analysis. 12 tumor bearing mice (3 mice each group) were sacrificed by standard decapitation 48 h later after US treatments. The tumors were harvested, fixed with formalin and embedded in paraffin. The tumor sections (8 μ m) were cut with a paraffin slicing machine (Leica, RM223, Germany) and stained with hematoxylin and eosin (H&E). For assessment of apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was carried out with an in situ Cell Death Detection Kit according to the manufacturer's protocol. Cell apoptosis index was determined by calculating the percentage of positively stained cells for all cells from six randomly chosen fields/sections at × 400 magnification. Statistical analysis. The results were expressed as the mean ± standard deviation (S.D). Comparisons between cell fluorescence intensities in the uptake experiments were compared using two-way repeated ANOVA test. Data analyses including cell viability, in vitro apoptosis/necrosis ratios, levels of ROS, in vivo fluorescence intensities and apoptotic index in tumor sections were performed with one-way ANOVA test. Tumor volumes and mouse body weights were compared using a Kruskal-Wallis test followed by the Manne-Whitney test. The differences were considered to be significant for * P < 0.05, and to be very significant for **P < 0.01.

Results
Cellular uptake of IR-780 iodide. The uptake of 4T1 cancer cells were investigated with flow cytometry and confocal laser scanning microscope. Results were showed in Fig. 2. Figure 2A demonstrated there were obvious dose-and time-dependence for cellular uptake of IR-780 iodide. At all three examined time points, the fluorescence intensities of the cells incubated with IR-780 iodide increased with an increase of IR-780 concentrations. After 1 h, the fluorescence intensity of the cells incubated with 10 μ M or 16 μ M of IR-780 iodide were 2.01-fold and 2.27-fold higher than that of the cells with 4 μ M of IR-780 iodide, respectively. Similar trends can be found in these cells incubated for 2 or 3 h. Also, stronger fluorescence intensities might be found in these cells with longer incubation time at the same dye dose, indicating the time-dependence of cellular uptake. Figure 2B showed the significant right shift of the fluorescence curves with the increasing dye concentrations after 1 h cell incubation. The results were further confirmed by a laser confocal microscopy, indicating the gradual increasing cellular uptake with the increase of IR-780 doses (Fig. 2C).
In vitro sonotoxic effect with IR-780 iodide on 4T1 cells. Figure 3A (Fig. 3G). These results indicated that IR-780 could act as a potential sonosensitizer for SDT.

Levels of ROS generation.
We further investigated whether IR-780 could induce the generation of ROS in the tumor cells after US irradiation. Figure 4A demonstrated that the tumor cells treated with IR-780 plus US caused significant increased level of 1 O 2 , with 1.55-fold, 2.29-fold and 2.91-fold higher fluorescence intensities than that of the tumor cells treated with IR-780, only US or PBS control, respectively (P < 0.01). Figure 4B demonstrated there had a significant increase of H 2 O 2 in the tumor cells treated with IR-780 plus US. There were 1.29-fold, 1.33-fold and 1.41-fold higher fluorescence intensities compared with the tumor cells treated with IR-780, only US or PBS control, respectively (P < 0.01). No significant increase of ⋅ OH levels was found in the tumor cells treated with IR-780 plus US (P > 0.05) (Fig. 4C).

Effect of active oxygen scavengers.
Since IR-780 could induce the generation of ROS in the tumor cells after US irradiation, we further used active oxygen scavengers to confirm the effects. The results were shown in Fig. 5. Obviously, cell damage by US alone was partly inhibited by histidine, but its enhancement by IR-780 was greatly inhibited (P < 0.01). SOD had no effect to inhibit the cell damage by US alone, while it could also decreased the cell damage by US + IR-780 (P < 0.01), which was similar with the effect of histidine. Mannitol had no effect in decreasing the cell damage of US with or without IR-780 (P > 0.05). In vivo imaging of IR-780 in tumor-bearing mice. In order to determine the optimal time for in vivo SDT using IR-780, we examined the tissue permeability and distribution of IR-780 in the tumor-bearing mice by a whole animal NIR imaging system. Figure 6A Fig. 6B, revealing that the fluorescence intensity in the tumors could almost achieve the maximum level after 1 h. The tumor localization of IR-780 was still dominant even if 48 h later (Fig. 6C,D). In vivo therapeutic efficacy of IR-780 plus US. Next, we tested the in vivo efficacy of IR-780 as a sonosensitizer. Figure 7A showed the representative tumor-bearing mice at day 0, day 11, day 23 and day 30. The tumor growth curves were presented in Fig. 7B. Obviously, mice received with IR-780 plus US showed a significant tumor growth inhibition (P < 0.01), with the mean tumor size of 40.7 ± 36.93 mm 3 at day 30. By contrast, there were not significant inhibition effects on tumor growth for IR-780 group or only US group (P > 0.05), with the mean tumor size of 973.06 ± 367.98 mm 3 or 921.62 ± 234.39 mm 3 at day 30, respectively. Figure 7C showed all of the tumors removed from four groups of tumor-bearing mice at day 30, demonstrating a dramatic decrease of tumor sizes when they received SDT with IR-780. No significant changes of body weight of mice were observed over the course of the mouse experiment (Fig. 7D). Additionally, we measured the surface temperature of the tumors through an electronic thermometer while the tumors were irradiated by US. It was demonstrated that the temperature of the tumors of both groups of animals receiving US did not have obvious increase, excluding the thermal effects by US irradiation in our experiments (data not shown).
Histological analyses. The tumor sections from mice after 48 h treatment were stained with H&E and examined under light microscope. A significantly increased degree of cell necrosis could be observed in the tumors receiving IR-780 and US (Fig. 8A, upper row). To elucidate whether SDT with IR-780 induce tumor cell apoptosis, TUNEL staining were performed. Results demonstrated that TUNEL-positive cells (green) were significantly more in the tumors treated using SDT with IR-780, compared with other groups (Fig. 8A, bottom row). Quantitatively, there were 50.9 ± 3.89% apoptotic tumor cells found in the tumors received IR-780 plus US. By contrast, there were 1.8 ± 0.77%, 1.85 ± 0.67% and 2.35 ± 0.57% apoptotic cells found in the tumors received PBS, only US or only IR-780, respectively (P < 0.01) (Fig. 8B).

Discussion
Currently, it is widely accepted that presence of potential sonosensitizers is one of the most essential factors in SDT. Identification and development of novel sonosensitizers are attracting more and more researchers' attention. In this study, we found the remarkable toxicity of SDT using IR-780 as a sonosensitizer against 4T1 breast cancer in vitro and in vivo. To our best knowledge, there is no literature reporting IR-780 agent as a sonosentizer in SDT. Our study provided evidences that IR-780 can function as a sonosentizer in SDT of breast tumor. In fact, some photosensitizers including porphyrin derivatives and phthalocyanins are also found to have great potentials    as sonosensitizers and attract extensive attentions 22 . Given the unique advantages of SDT over PDT, there are enough reasons for us to believe that IR-780 can have a promising application in tumor treatment.
In our study, 4T1 breast cancer cells demonstrated excellent uptake efficiency for IR-780 iodide (Fig. 2). It is easy to understand when considering the fact that the lipophilic property of this dye may favor it to effectively cross the membrane into the tumor cells. Also, we found that the free IR-780 iodide and only US had some anti-proliferative activity on 4T1 breast cancer cells (Fig. 3A), demonstrating a proper dye dose and US energy should be optimized for future clinical utilities. Also, several approaches, such as encapsulation of this dye with biocompatible nanomaterials or utilization of local intratumoral administration, may decrease the potential cytotoxicity of IR-780 dye to health cells. It was demonstrated the cytotoxicity from the non-thermal US mainly result from the mechanical stress or sonochemical effects of acoustic cavitation 23 . In our study, SDT with IR-780 mainly led to cell necrosis and late apoptosis (Fig. 3B-G). It is known that PDT can induce apoptosis and necrosis, and necrotic response may be the major cell death pathway in high-dose photosensitizer with PDT 24 . This also can be expected for SDT with IR-780. In the Tsuru's report, it was proved that SDT with DEG mainly led to necrosis instead of apoptosis 25 . Indeed, various kinds of factors, such as sonosensitizers, tumor cell lines, and US parameters, may affect the ratios of apoptosis or necrosis of tumor cells in SDT 26 .
The generation of ROS plays an important role in SDT of tumor treatment. Our data showed that 1 O 2 and H 2 O 2 , but not ⋅ OH were found to be an increased generation after 4T1 cancer cells treated with IR-780 and US (Fig. 4A,B). This was confirmed by the sonodynamic cell damage experiments, in which the sonodynamic cell damage can be inhibited by histidine and SOD, but not by mannitol (Fig. 5). The species of ROS induced by SDT were involved in the kinds of sonosensitizers 27 . Previous studies revealed that singlet oxygen was a major source of ROS from porphyrin-derived sensitizers in both PDT and SDT 28,29 . Our result was consistent with the SDT with porphyrins 30,31 . Thus, a possible mechanism of IR-780 for SDT may be speculated as follows: IR-780 can be uptaken by the tumor cells. Upon receiving US irradiation, IR-780 in the tumor cells would be activated and changed into the excited state from the ground state. Due to the instability of the excited-state IR-780, they would trend to return to the ground state and to release the US energy. Oxygen molecules and H 2 O can acquire the released energy and turn into 1 O 2 and H 2 O 2 . With the increase of the concentrations of 1 O 2 and H 2 O 2 , the tumor cells would be progressively damaged through oxidative stress-induced cell death pathways (Fig. 9). In fact, the specific mechanisms of SDT are dependent on various factors, including tumor cell models, experimental systems utilized, the type of sonosensitizers and the US exposure parameters (such as frequency and intensity) 32 . It is also difficult to give a universal mechanism of SDT. Our study, at least partly, demonstrated that SDT with IR-780 can kill 4T1 tumor cells through increasing the level of 1 O 2 and H 2 O 2 in these tumor cells.
In this study, we further investigated the efficacy of SDT with IR-780 and demonstrated the great tumor growth inhibition in vivo xenografts of 4T1 cancer cells (Fig. 7). An obvious tumor necrosis and more apoptotic tumor cells could be observed in the H&E-or TUNEL-stained microscopic section of tumors received SDT with IR-780 (Fig. 8). The increased apoptosis may contribute to the generation of ROS in the process of IR-780 exposure to US. No obvious adverse effects were observed during our tumor treatments. Our data showed for the first time that SDT with IR-780 significantly inhibited the tumor growth with increased apoptosis and necrosis. Thus provides a promising SDT agent for tumor treatment with minimal side effects.