Discovery of a pyridophenoselenazinium-based photosensitizer with high photodynamic efficacy against breast cancer cells

Abstract

Development of efficient photosensitizers with minimal side effects is highly desirable for photodynamic therapy. Reported herein is the discovery of a novel pyridophenoselenazinium-based NIR-I photosensitizer, termed PPSe, that efficiently generated both type I and II reactive oxygen species (ROS) upon appropriate light irradiation. PPSe exhibited potent phototoxicity as well as excellent phototherapeutic indices against several breast cancer cell lines. PPSe induced DNA damage and breast cancer cell apoptosis via photo-triggered intracellular ROS generation.

Main article text

1. INTRODUCTION

Breast cancer (BC) is the most common cancer among women, and one of the leading causes of female morbidity and mortality, with 2.26 million new cases and 680,000 deaths in 2020 worldwide [1–3]. Thus, breast cancer remains a major public health burden. Current breast cancer therapies mainly rely on surgery, radiotherapy, chemotherapy, and targeted therapy; however, these strategies are limited by physical pain, fatal side effects, and drug resistance [4–6]. The situation for aggressive triple negative breast cancer (TNBC) is even worse due to its lack of progesterone receptors (PRs), human epidermal growth factor receptor 2 (HER2), and estrogen receptors (ERs) [7, 8]. Therefore, it is of great importance to develop more efficient and less toxic therapies for breast cancer treatment, especially TNBC.

In recent years, photodynamic therapy (PDT) has attracted much interest for breast cancer treatment owing to its low systemic toxicity, minimal invasiveness, negligible drug resistance, and cost effectiveness [9–11]. Typically, PDT utilizes a combination of photosensitizer, light, and oxygen to destroy cancer cells. An ideal photosensitizer should be non-toxic, but possess significant ROS generation capacity upon light irradiation [12]. Photosensitizers are mainly divided into type I and II based on the mechanism of ROS generation mechanism. Upon photoirradiation, type I photosensitizers produce radical reactive oxygen species, such as superoxide anion radical (O₂ ^•−) and hydroxyl radical (OH^•), via an electron transfer process, while type II photosensitizers convert non-toxic molecular oxygen into highly toxic singlet oxygen (¹O₂) via an energy transfer photoreaction [13, 14]. Type I photosensitizers are thought to be more efficient against solid tumors because the electron transfer pathway is less O₂-dependent. A number of photosensitizers have been used in clinical applications over the past 30 years, such as porphyrin, chlorin, phthalocyanine, and indocyanine green; however, most photosensitizers used clinically are type II, which is highly O₂-dependent. In addition, current photosensitizers exhibit high dark toxicity, a limited penetration depth, and are challenging to synthesize [12, 15]. Therefore, new photosensitizers with excellent efficacy and minimal side effects for photodynamic cancer therapy, especially type I photosensitizers, are still in high demand.

Methylene blue (MB) is an FDA-approved phenothiazinium-based drug for the treatment of methemoglobinemia. Methylene blue has an excellent safety profile and has been shown to be an antitumor photosensitizer; however, the redox-active property of methylene blue results in poor intracellular ROS production capacity and limited therapeutic efficacy. In this work we report the discovery of a novel pyridophenoselenazinium-based NIR-I photosensitizer (PPSe) derived from methylene blue that exhibited potent phototoxicity along with excellent phototherapeutic indices against several breast cancer cell lines, including MDA-MB-231, MDA-MB-468, MCF-7, and 4T1 cells.

2. MATERIALS AND METHODS

2.1 Determination of singlet oxygen quantum yield

Singlet oxygen production was evaluated with 1,3-diphenylisobenzofuran (DPBF). The singlet oxygen generation of PPSe was measured in ethanol solutions and MB was used as a standard. The absorbance of DPBF at 410 nm was adjusted to approximately 1.0, and the absorbance of PPSe and MB at 660 nm was adjusted between 0.2 and 0.4. Then, the absorption spectrum was measured after light irradiation (660 nm, 2 mW·cm^-2) for 0, 10, 20, 30, 40, 50, 60, 70, and 80 s. DPBF solution was used as the control. The singlet oxygen quantum yield was monitored by measuring the reduction in the absorbance intensity of DPBF at 410 nm using a microplate reader (Tecan, Switzerland). The values of singlet oxygen, Φ _Δ, were calculated using the following equation:

               

where, Φ _Δ represents the singlet oxygen quantum yield of PPSe, Φ _MB is the singlet oxygen quantum yield of MB (0.52 in ethanol), k represents the slope of the decrease in DPBF absorbance at 410 nm with the addition of irradiation time, and F is the correction factor (F = 1 − 10^−OD). OD represents the absorbance of the solution at 660 nm.

2.2 Superoxide anion radical (O₂ ^•−) detection

To determine O₂ ^•− generation, dihydrorhodamine 123 (DHR123) was used as a superoxide anion radical probe. PPSe was added to a DHR123 aqueous solution at the same concentration (5 μM). The solutions were exposed to 660 nm irradiation (10 mW/cm²) for different times. DHR123 without PPSe was used as a control. The fluorescence intensities of solutions were measured using a spectrofluorometer and the excitation wavelength was 500 nm.

2.3 Cell incubation

Human breast cancer cell lines (MDA-MB-231, MCF-7, MDA-MB-468, and MDA-MB-453) and mouse breast cancer 4T1 cells were obtained from Shandong University (Jinan, China). The human breast cancer cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% double antibiotics (penicillin/streptomycin; Gibco, Grand Island, NY, USA). And the mouse breast cancer 4T1 cell was cultured in RPMI-1640 cell medium supplemented with 10% FBS and 1% double antibiotics (penicillin/streptomycin). All the cells were incubated in a 5% CO₂ humidified incubator at 37°C.

2.4 Cellular uptake

Cellular uptake efficiency of PPSe was determined using CytoFLEX S Flow Cytometry (Beckman Coulter, Brea, California, USA). 4T1 cells were incubated with PPSe (5 μM) for the following lengths of time: 30 min; 1 h; 3 h; 6 h; and 9 h. Then, the cells were harvested and re-suspended with phosphate buffer solution and analyzed by flow cytometry. The excitation wavelength was 638 nm and the emission collection wavelength was 712 ± 25 nm.

2.5 Intracellular ROS generation

The in vitro ROS generation of PPSe in 4T1 cells was assessed using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as a probe. The 4T1 cells were incubated with PPSe (0.25 μM) for 2 h at 37°C, then co-incubated with DCFH-DA (10 μM, λex = 488 nm, λem = 525 nm) for 30 min at 37 °C in the dark. 4T1 cells were washed after incubation, followed by irradiation (660 nm, 20 mW/cm², 10 min). The cells were carefully collected and analyzed immediately using flow cytometry (Beckman). Cells in the medium without compound were used as controls, and the experiments were repeated three times.

2.6 Cytotoxicity test

The cytotoxicity of PPSe was carried out using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay. MDA-MB-231, MCF-7, MDA-MB-468, MDA-MB-453, and 4T1 cells were seeded on a 96-well plate at a density of 5000 cells/well for a 24-h incubation. The photosensitizer, PPSe, was diluted into different concentrations with medium. The cells were incubated with different concentrations of PPSe for 6 h, then irradiated with a 660-nm LED lamp (20 mW/cm², 10 min). After a 24-h incubation, the CCK-8 stop solution (10 μL) was added to each well and the cell viabilities were measured. The same concentrations of PPSe were added to cancer cells for 24 h in darkness to evaluate dark cytotoxicity. After CCK-8 treatment, the absorbance of each at 450 nm was measured using a microplate reader (Tecan) and IC₅₀ values were calculated accordingly. The experiments were repeated three times. Cell viability rates (%) and IC₅₀ values were analyzed using GraphPad Prism 9.0.7.

2.7 Calcein AM/PI assay

Calcein-AM and propidium iodide (PI) were used to study live/dead cell co-staining. 4T1 cells were seeded in 35-mm plates and cultured for 24 h. The cells were incubated with PPSe (0.25 and 0.5 μM) for 6 h, then treated with or without irradiation (660 nm, 20 mW/cm², 10 min). Cell groups with or without irradiation were used as controls. After a 12-h incubation, the cells were first washed with phosphate buffer solution, then co-stained with calcein AM/PI for 30 min at 37 °C in the dark. Finally, all the groups of cells were imaged with a confocal laser scanning microscope (Zeiss, Germany).

2.8 Apoptosis assays

Cell apoptosis was analyzed using an Annexin V-FITC and PI apoptosis detection kit (Beyotime Biotechnology, Shanghai, China). 4T1 cells were cultured in 6-well Corning plates and incubated with PPSe (0.25 and 0.5 μM) at 37 °C for 6 h, then treated with or without irradiation (660 nm, 20 mW/cm², 10 min). The 4T1 cells were incubated for 24 h after irradiation, collected, and stained with Annexin V-FITC and PI at room temperature for 30 min to analyze cell apoptosis. Cell apoptosis was detected by flow cytometry (Beckman), and the percentage of apoptotic cells was analyzed using FlowJo.

2.9 DNA damage and apoptosis (Western blotting)

The 4T1 cells were seeded in 6-well Corning plates at a density of 2×10⁵ cells/well and incubated for 24 h. Then, the cells were incubated with PPSe (0.25 μM) for 6 h and irradiated with a 660-nm LED lamp (20 mW/cm², 10 min). Cells were collected after incubation for 24 h, then lysed using RIPA lysis solution (Beyotime Biotechnology) with the addition of protease/phosphatase inhibitors for 30 min on ice. After calibrating the concentration of protein, equal amounts of protein were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose (NC) filter membranes. The primary antibodies were mouse monoclonal caspase-3 (Proteintech, Wuhan, China) γ-H2A.X (Abcam, Cambridge, UK), and β-actin (Proteintech, Wuhan, China). Anti-mouse IgG HRP-conjugated antibody (Proteintech, Wuhan, China) was used as a secondary antibody. An ECL detection system (Tanon, Shanghai, China) was used to visualize the specific protein bands. The target protein levels were semi-quantitatively analyzed using Image J software.

2.10 Statistical analysis

The statistical analysis between groups was performed with one-way analysis of variance (ANOVA) and Student’s t-test using GraphPad Prism 9.0.7. software. The results are expressed as the mean ± standard deviation (SD). A p value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001).

3. RESULTS AND DISCUSSION

The photosensitizer, PPSe, was synthesized through redox-neutral annulation between readily accessible 3,3’-diselanediylbis (N, N-dimethyl-4-nitrosoaniline) and 4-[(quinolin-8-ylamino) methyl] benzoic acid, as shown in Scheme S1 .

The optical properties of PPSe were first analyzed using UV-vis absorption and fluorescence spectra. As shown in Figure 1b , the maximum absorption of PPSe centered at approximately 660 nm. Upon 660 nm light excitation, PPSe exhibited NIR-I emission with a maximum peak at approximately 690 nm.

Figure 1 |

(a) The structure of the photosensitizer, PPSe. (b) UV-vis and fluorescence spectra of PPSe in ethanol. (c-d) Absorption changes of the DPBF/PPSe under 660-nm LED light irradiation (2 mW/cm²) in ethanol solution. (e) O₂ ^•− detection using the DHR123 (5 μM) assay for PPSe (5 μM) in aqueous solution upon irradiation (660 nm, 10 mW/cm²). (f) Fluorescence response of DHR123 for PPSe (5 μM) and DHR123 (5 μM) in aqueous solution.

The ROS generation capacity of PPSe was then evaluated because the ROS generation capacity represents the photodynamic efficacy of a photosensitizer. To determine whether PPSe generated ¹O₂, DPBF was used as the ¹O₂ capture probe and MB (Φ _Δ = 0.52 in ethanol) was used as a reference ( Figures 1c, d and S1 ). As the irradiation time increased, the absorbance of the DPBF/PPSe ethanol solution decreased under 660 nm light irradiation (2 mW/cm²). The singlet oxygen quantum yield of PPSe in ethanol was determined to be 0.62, indicating that PPSe had strong singlet oxygen generation efficiency. To characterize the superoxide anion radical generation ability of PPSe in the aqueous solution upon irradiation, a DHR123 probe was used as the superoxide anion radical (O₂ ^•−) indicator. There was no obvious fluorescence change in the DHR123 alone group under irradiation (660 nm, 10 mW/cm²), but significant fluorescence enhancement of the PPSe (5 μM) and DHR123 (5 μM) group was observed, indicating O₂ ^•− production ( Figure 1e, 1f ). Taken together, PPSe efficiently produced ROS through electron and energy transfer processes. Thus, PPSe is a type I/II combined photosensitizer.

To investigate the cellular uptake efficiency of PPSe, flow cytometry was used to monitor the changes in fluorescence intensity among living cells. The 4T1 cells were treated with PPSe (5 μM) at the following times: 30 min; 1 h; 3 h; 6 h; and 9 h. As shown in Figure 2a and 2b , a right shift of the chromatogram represents increased fluorescent intensity, which can indicate increased cellular uptake. It was observed that the cellular uptake of PPSe displayed a gradual increase over time, with saturation at approximately 6 h. Overall, the cellular uptake experiment showed that PPSe was efficiently taken up by breast cancer cells.

Figure 2 |

(a-b) Flow cytometry analysis and the curve of median fluorescence intensity of cellular uptake of PPSe (5 μM) after incubation for different time periods. (c-d) Flow cytometry analysis and the curve of median fluorescence intensity of ROS generation in 4T1 cells treated with PPSe (0.25 μM) with or without light irradiation (660 nm, 12 J/cm²). Results are expressed as mean ± SD (n=3), ***P < 0.001 vs the PBS plus light group.

Because PPSe exhibited PDT potential by generating ¹O₂ and O₂ ^•− as discussed above, the intracellular ROS produced by PPSe in living cells were further measured using DCFH-DA as a fluorescent probe. The breast cancer 4T1 cells were treated with PPSe (0.25 μM) for 6 h, followed by incubation with the DCFH-DA (10 μM) probe for 30 min. As shown in Figure 2c and 2d , there were no apparent differences in the PBS groups with or without irradiation. The intracellular ROS level in the PPSe-treated cells was negligible in the dark, but significantly increased upon 660 nm irradiation (20 mW/cm², 10 min). The results indicated that PPSe efficiently induced ROS generation under light irradiation and might have potent phototoxicity.

To assess the photodynamic effect of PPSe against breast cancer cells, the cytotoxicity of PPSe against 4T1 cells was investigated using the CCK-8 assay. As depicted in Figure 3a , the cell viabilities of 4T1 cells incubated with different concentrations of PPSe and exposed to different light doses (660 nm, 4 J/cm², 8 J/cm², 12 J/cm²) were evaluated. The results indicated that the phototoxicity of PPSe increased in a dose-dependent manner within the investigated light dose ranges, and PPSe exhibited the best photodynamic efficacy under the irradiation of 12 J/cm² (20 mW/cm², 10 min). Notably, PPSe exhibited excellent cytotoxicity against 4T1 cell lines with an IC₅₀ value of 170.2 ± 9.6 nM upon 660 nm light irradiation (12 J/cm²), as shown in Figure 3b . Furthermore, the photodynamic effect of PPSe under hypoxia was also investigated. Because PPSe mainly generated ¹O₂ following a type II pathway, PPSe exhibited poor phototherapeutic efficacy without a dose-dependent fashion under 1% O₂ conditions ( Figure S5 ). It is noteworthy that PPSe exhibited no significant dark toxicity within the tested concentrations, which indicated that PPSe has an excellent phototherapeutic index (> 58) against 4T1 cells.

Figure 3 |

(a) Cell viabilities of 4T1 cells incubated with various concentrations of PPSe after exposure to different light doses (660 nm). (b) Cell viabilities of 4T1 cells incubated with various concentrations of PPSe with or without irradiation (660 nm, 12 J/cm²). (c) Fluorescence images of Calcein-AM and PI co-stained 4T1 cells treated with PPSe with or without irradiation, the irradiation is 660 nm, 12 J/cm². Scale bar: 100 μm.

To visually confirm the cytotoxicity of PPSe, the photodynamic effect of PPSe toward cancer cells was further evaluated by the live/dead cell co-staining assay using PI and calcein-AM. Calcein-AM stains live cells to emit green fluorescence, whereas PI stains dead cells to emit red fluorescence. As shown in Figure 3c , almost no cells were dead in the PBS control groups or the groups treated with PPSe (0.25 and 0.5 μM) without light irradiation, in which a large percentage of cells were stained green by calcein-AM. By contrast, the cancer cells treated with PPSe were significantly killed in a concentration-dependent manner upon light irradiation. As shown in Figure 3c , nearly one-half of the 4T1 cells were killed when incubated with 0.25 μM PPSe plus 660 nm light irradiation (20 mW/cm², 10 min). In addition, significant red fluorescence was observed after treating 4T1 cells with 0.5 μM PPSe plus light irradiation, indicating that nearly all the cells were efficiently killed, which correlated well with the cell viability assay findings.

To further evaluate the cytotoxicity of PPSe against human breast cancer cells, the viability of MDA-MB-231, MCF-7, MDA-MB-468, and MDA-MB-453 cells was also determined using the CCK-8 assay. As shown in Figure 4 and Table S1 , there were slight differences among the four breast cancer cell lines. The IC₅₀ values of PPSe against the MDA-MB-231, MCF-7, MDA-MB-468, and MDA-MB-453 human breast cancer cell lines under 660-nm light irradiation (20 mW/cm², 10 min) conditions were 0.16 ± 0.010, 0.71 ± 0.061, 0.12 ± 0.014, and 0.29 ± 0.012 μM, respectively, which were similar to the 4T1 cell results. The dark toxicity of PPSe was also determined under the same experimental conditions except for light irradiation. It was shown that PPSe exhibited weak dark toxicity within the tested concentrations against the breast cancer cell lines. It should be noted that the phototherapeutic index of PPSe against MDA-MB-231 cells was excellent (>125).

Figure 4 |

MDA-MB-231 (a) MCF-7 (b) MDA-MB-468 (c) and MDA-MB-453 (d) cell viabilities after incubation with various concentrations of PPSe with or without irradiation (660 nm, 12 J/cm²).

The flow cytometry assays with Annexin V-FITC/PI staining were performed to evaluate the apoptotic effects of PPSe on 4T1 cells. As illustrated in Figure 5a and 5b , the percentage of apoptotic cells was significantly increased in the PPSe plus irradiation (660 nm, 20 mW/cm², 10 min) groups compared with the PPSe alone groups. The cell apoptotic rates in the PPSe (0.25 μM) alone group and the PPSe (0.25 μM) plus irradiation group were 2.31% and 17.32%, respectively. When the 4T1 cells were incubated with 0.5 μM PPSe plus irradiation, the cell apoptotic rate was significantly increased to 32.51%. By contrast, there was no significant change in the cell apoptosis rate of the PBS groups whether irradiated or not. Taken together, PPSe efficiently induced 4T1 cell apoptosis under light irradiation and the percentage of apoptotic cells increased as the concentration of PPSe increased, which is consistent with the cytotoxicity of PPSe.

Figure 5 |

(a-b) Effects of PPSe on cell apoptosis in 4T1 cells. Results are expressed as mean ± SD (n=3), **P < 0.01 and ***P < 0.001 all vs the PBS plus light group. (c) Western blot assay of DNA damage-relevant protein γ-H2A.X and apoptosis-relevant protein cleaved caspase-3 protein in 4T1 cells, β-actin utilized as an internal loading control. (d-e) Quantitative protein expression of cleaved caspase-3 and γ-H2A.X. All results are expressed as mean ± SD (n=3), ***P < 0.001 vs the PBS plus light group.

To further investigate the mechanism of cell death induced by PPSe under light irradiation, we performed Western blot assays to monitor DNA damage and apoptosis in 4T1 cells. As shown in Figure 5c , the expression of cleaved-caspase-3 and γ-H2A.X increased significantly in the PPSe (0.5 μM) plus light irradiation (660 nm, 20 mW/cm², 10 min) group, but not in the PPSe alone (0.5 μM) and PBS control groups. These results indicated that the combination of PPSe and light irradiation led to efficient DNA damage and induced apoptosis.

4. CONCLUSIONS

In summary, we have described the discovery and characterization of a new NIR-I photosensitizer (PPSe) that efficiently generated ROS via a type I and II combined pathway in the presence of light. PPSe exhibited potent phototoxicity against five breast cancer cell lines. It is noteworthy that the phototherapeutic index of PPSe against MDA-MB-231 cells was excellent (>125). Cell death mechanism studies indicated that PPSe induced DNA damage and cell apoptosis in the presence of light. We hope that PPSe will be considered suitable for more applications in PDT.

Supplementary Material

Supplementary Material can be downloaded here

CONFLICTS OF INTEREST

The authors declare no competing financial interest.

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Graphic abstract

Highlights

• A novel pyridophenoselenazinium-based NIR-I photosensitizer (PPSe) was developed.

• PPSe efficiently generated both type I and II ROS upon appropriate light irradiation and exhibited high photodynamic efficacy against breast cancer cells.

• PPSe induced DNA damage and breast cancer cell apoptosis.

In brief

A novel pyridophenoselenazinium-based NIR-I photosensitizer (PPSe) with potent phototoxicity as well as excellent phototherapeutic indices against breast cancer cells was developed.