Microenvironment-triggered dual-activation of a photosensitizer- fluorophore conjugate for tumor specific imaging and photodynamic therapy

Photodynamic therapy is attracting increasing attention, but how to increase its tumor-specificity remains a daunting challenge. Herein we report a theranostic probe (azo-PDT) that integrates pyropheophorbide α as a photosensitizer and a NIR fluorophore for tumor imaging. The two functionalities are linked with a hypoxic-sensitive azo group. Under normal conditions, both the phototoxicity of the photosensitizer and the fluorescence of the fluorophore are inhibited. While under hypoxic condition, the reductive cleavage of the azo group will restore both functions, leading to tumor specific fluorescence imaging and phototoxicity. The results showed that azo-PDT selectively images BEL-7402 cells under hypoxia, and simultaneously inhibits BEL-7402 cell proliferation after near-infrared irradiation under hypoxia, while little effect on BEL-7402 cell viability was observed under normoxia. These results confirm the feasibility of our design strategy to improve the tumor-targeting ability of photodynamic therapy, and presents azo-PDT probe as a promising dual functional agent.

Scientific RepoRtS | (2020) 10:12127 | https://doi.org/10.1038/s41598-020-68847-w www.nature.com/scientificreports/ those of normal tissues. The features of these tumor microenvironments have been widely used for the design of tumor-targeted therapy 4,5 . Hypoxia is an important microenvironmental factor in cancer 6 . Therapies that are originally inert but sensitive to hypoxia may provide tumor-specificity. Taking advantage of hypoxia in solid tumors, many efforts have been devoted to develop tumor-targeted therapeutic or imaging agents [7][8][9][10][11][12][13][14][15][16] . Nagano and Urano et al. first reported that the azo group is sensitive toward hypoxia and may be employed to design fluorogenic probes for the detection of hypoxia 17 . Furthermore, they used the azo group as a quencher to design hypoxia-activated photosensitizers 12 . Recently, the groups of Tan, Fang, and Zhao collaborated to successfully design a hypoxia-activated aptamer for cancer imaging with improved specificity 13 . Based on hypoxia, Pu et al. successfully activated a prodrug of the chemotherapeutic drug Br-IPM 14 . All these results prove the advantage of the hypoxic tumor environment as a target to design tumor-specific imaging or therapy modalities. While many studies have been reported on hypoxia-activated therapeutic or imaging agents, hypoxia-dependent dual activation for simultaneous tumor imaging and photodynamic therapy has not been investigated to date, to the best of our knowledge.
To design bifunctional probes for hypoxia-dependent tumor imaging and photodynamic therapy, we started by interrogating the mechanism by which photosensitizers convert light energy into singlet oxygen. When a chromophore absorbs a photon, it is promoted to an excited state. The excited chromophore can lose energy by populating the first excited singlet state via internal conversion followed by rapid relaxation back to the ground state. However, for a photosensitizer, the excited singlet state electron is easily undergoing spin inversion to populate the first excited triplet state at lower energies via intersystem crossing. This triplet state readily interacts with ground-state molecular oxygen ( 3 O 2 ), which is a triplet state, leading to the production of radicals and reactive oxygen species 1 . Obviously, photochemical processes catalyzed by photosensitizers rely on the population of their triplet state. We hypothesized that inhibition of this state with a hypoxia-sensitive trigger may block the photochemical reaction under normoxia. Under hypoxia, the hypoxia-sensitive trigger switches on the photosensitizers and makes them ready for the photochemical reaction, leading to a hypoxia-specific photodynamic effect. www.nature.com/scientificreports/ Fluorescence resonance energy transfer (FRET) is the absorption of the energy of a donor in its excited state by an acceptor structure, which may be employed to absorb the energy of an excited photosensitizer to inhibit the population in its excited triplet state. Herein, utilizing the FRET mechanism, we conjugated a photosensitizer (as energy donor) and a near-infrared fluorophore (as energy acceptor) with an azo group as hypoxia trigger, to successfully design a new chemical entity (azo-PDT) that can not only detect solid tumors based on its hypoxiaactivated fluorogenic response but also show hypoxia-specific phototoxicity for the ablation of tumor cells.

Results and discussion
Design and synthesis. We chose pyropheophorbide α (Pyro), which is an analog of Photofrin, as the photosensitizer for our proof-of-concept study due to its easy availability. For efficient FRET, donor emission and acceptor absorption should be adjacent and overlap 18 . Given the extremely low emission of Pyro around 700-800 nm, we reasoned that the Si-rhodamine fluorescent dye SiR-665 could act as the energy acceptor due to its absorption around 600-750 nm 19 . Another advantage of SiR-665 is its strong NIR II emission, which qualifies it as a fluorophore for tumor imaging. We designed the entity azo-PDT, which uses the azo group as hypoxia-specific trigger and also as linker between the photosensitizer Pyro and the fluorophore SiR-665. We reasoned that due to the FRET effect between Pyro and SiR-665, no photodynamic effect should be observed, not even under irradiation. Due to the azo-caused fluorescence quenching of SiR-665 17 , the probe should be non-emissive. Hypoxia will lead to the reductive cleavage of the azo group 17 , effectively separating the two functional moieties and, thus, activating both activity (Fig. 1b).
Procedures for the synthesis of azo-PDT are outlined in Fig. 2 and detailed in Scheme S1. Briefly, starting with 3-bromoaniline, we obtained comp. 3 (Table 1). Then, the singlet oxygen quantum yield (Φ( 1 O 2 )) of azo-PDT was quantitatively measured in comparison with that of Pyro according to a reported method 20 Table 1). The significantly lower Φ( 1 O 2 ) value of azo-PDT compared with Pyro suggests effective quenching of the photoreactivity of azo-PDT due to the efficient FRET process between Pyro and SiR-665. This is in accord with the photophysical data, which showed partial overlap between the emission of Pyro and the absorption of SiR-665 (Fig. 3b).
After confirming the effective quenching of the 1 O 2 -production ability of azo-PDT, we measured its fluorescence properties. As shown in Fig. 3b, azo-PDT is almost non-fluorescent, although SiR-665 is highly emissive. This finding indicates that the fluorescence of SiR-665 fluorophore is significantly quenched by the azo group.

Hypoxia-activated fluorescence of azo-PDT in aqueous solution.
After confirming the effective inhibition of the 1 O 2 -generation ability of azo-PDT and quenching of its fluorescence emission, we tested if reductive cleavage of the azo group restores both activities. For this purpose, the biological reductive hypoxic environment was mimicked with mouse liver microsomes 17 . After treating azo-PDT (5 μM) with mouse liver microsomes (75 μg/mL) in the presence of NADPH (50 μM) as a reductase cofactor, an increase of the probe fluorescence was observed, which intensified with the proceeding of the incubation (Fig. 4a).
We also checked if other biological species commonly found in live cells induce the switch-on of the fluorescence of azo-PDT. For this purpose, azo-PDT in PBS was treated with various analytes for 1 h, and then its fluorescence was observed. As shown in Fig. 4b, microsomes were the only analytes to induce its fluorescence, suggesting that azo-PDT is specifically activated by the biological hypoxic environment. This is highly advantageous, as this suggests that azo-PDT should have a tumor-specific photodynamic effect.
Furthermore, we also confirmed the reductive cleavage of the azo bond in azo-PDT to yield the free amino-SiR-665 by LC-MS analysis (Fig. S1), supporting our design rationale.

Hypoxia-activated fluorescence and photoreactivity of azo-PDT in live cells. Having confirmed
the hypoxia-activated fluorescence of azo-PDT in aqueous solution, we studied if azo-PDT retains this feature in live cells for hypoxia-specific imaging and photodynamic ablation. For this purpose, we first screened a panel of cells to select the most sensitive one and applied azo-PDT to these cells under hypoxia (1% O 2 ). All cells demonstrated higher intracellular probe fluorescence under hypoxia than under normoxia (Fig. S2), suggesting that azo-PDT may be activated under hypoxia in all tested cell lines. It is noteworthy that BEL-7402 cells showed the most dramatic intracellular fluorescence increase under hypoxia, suggesting that azo-PDT is more sensitive in BEL-7402 cells, which were thus subject of further research.
First, the influence of the hypoxia incubation time on the activation of azo-PDT was studied by detecting the fluorescence switch-on effect. For this purpose, BEL-7402 cells were incubated with azo-PDT (5 μM) under hypoxia or normoxia for various times. While no significant time-dependent increase of the intracellular fluorescence was observed in the normoxia group, the intracellular azo-PDT fluorescence increased with increasing incubation time in the hypoxia group. These results suggest that, under hypoxia, the azo group in azo-PDT is reductively cleaved, which occurs gradually with increasing incubation times (Fig. 5).
In the following, we optimized the working concentration of azo-PDT to stain cells. For this purpose, BEL-7402 cells were incubated with various concentrations of azo-PDT under normoxia or hypoxia for 6 h. Cells under normoxia demonstrated negligible intracellular azo-PDT fluorescence, indicating that the fluorescence of azo-PDT is quenched, while the intracellular fluorescence of cells under hypoxia depended on the azo-PDT concentration (Fig. S3). Our results showed that an azo-PDT concentration of 2.5 μM was sufficient to yield significant intracellular fluorescence under hypoxia. Therefore, we chose this azo-PDT working concentration Table 1. Photophysical and photochemical parameters of azo-PDT, methylene blue, SiR-665 and pyro. a The data were obtained in PBS. b The data were measured in EtOH. c Φ Δ : singlet oxygen quantum yield. For methylene blue, Φ Δ = 0.49. "/" means not detectable, and "-" means not detected.   (Fig. S4).
After confirming the hypoxia-dependent activation of the fluorescence of azo-PDT in BEL-7402 cells, we tested if hypoxia also restores the 1 O 2 -generation ability of azo-PDT in BEL-7402 cells. BEL-7402 cells were incubated with azo-PDT under normoxia or hypoxia for 6 h and then irradiated with LED light at 670 nm for 20 min to induce the production of 1 O 2 . Then, the cells were incubated without irradiation for another 24 h, followed by SRB assay and cck-8 assay to measure the cell viability using Pyro as a positive control. While the Pyro group showed an irradiation-dependent cell ablation effect under both normoxia and hypoxia, low concentrations of azo-PDT only ablated the cell viability under hypoxia after irradiation, suggesting its hypoxia specificity (Fig. S5). The cytotoxicity of azo-PDT (2.5 μM) under normoxia or hypoxia, and with or without irridaition was summarized in Fig. 6 with pyro as a positive control. In contrast to pyro which shows photoirradiation-dependent cytotoxicity either under normoxia or hypoxia, azo-PDT showed potent cytotoxicity only under hypoxia when photo-irradiated. This observation suggests that the cell ablation effect of azo-PDT relies on both photo-irradiation and hypoxia activation, which confirms the success of our designed probe. To make further confirmation that the hypoxia-photo-irradiation-dependent cytotoxicity of azo-PDT is indeed due to its induction of ROS generation, we checked the cellular ROS levels by staining cells with 2′, 7′-dichlorofluorescin  In summary, utilizing the resonant energy transfer between pyropheophorbide α and the quenched fluorophore SiR-665, we have developed a "pro-photosensitizer" that is activated under hypoxia in tumor cells. Due to the energy transfer between the photosensitizer and the quenched fluorophore, the pro-photosensitizer does not generate singlet oxygen to damage cells under normoxia. Under hypoxia, the azo group undergoes reductive cleavage, effectively separating the photosensitizer and fluorophore and, consequently disrupting the energy transfer between these groups and restoring the fluorescence of the fluorophore as well as the photoactivity of the photosensitizer. Applying this strategy, tumor-selective imaging and photodynamic therapy may be realized. We validated the feasibility of this strategy in live BEL-7402 cells. We have shown that 2.5 μM azo-PDT achieved real-time imaging and showed selective killing activity. Overall, azo-PDT may be a promising dual-functional imaging tool to detect cancer hypoxia as well as to achieve tumor-specific cancer therapy. Furthermore, the design strategy introduced in this study may be inspiring for future probe design.   cell culture. Different cell lines. 1.5 × 10 5 cells were seeded onto 3.5 cm Petri dishes and cultured in corresponding medium with 10% (v/v) FBS and incubated for 12 h. The tested compounds were diluted to 5 μM, then the cells were incubated for 1 h under an atmosphere of 5% CO 2 in air, thereafter the cells were incubated for 6 h under an atmosphere of 5% CO 2 and 1% O 2 (hypoxia) or for 6 h under an atmosphere of 5% CO 2 in air (normoxia). The medium was removed and washed with fresh medium. Confocal Fluorescence images were then obtained on Olympus IX83-FV3000.

Methods
Different hypoxia hours. 1.5 × 10 5 cells were seeded onto 3.5 cm Petri dishes and cultured in RPMI 1640 medium with 10% (v/v) FBS and incubated for 12 h. The tested compounds were diluted to 5 μM, then the cells were incubated for 1 h under an atmosphere of 5% CO 2 in air, thereafter the cells were incubated for different hours under an atmosphere of 5% CO 2 and 1% O 2 . The medium was removed and washed with fresh medium. Confocal Fluorescence images were then obtained on Olympus IX83-FV3000.
Different concentrations. 1.5 × 10 5 cells were seeded onto 3.5 cm Petri dishes and cultured in RPMI 1640 medium with 10% (v/v) FBS and incubated for 12 h. The tested compounds were diluted to different final concentrations, then the cells were incubated for 1 h under an atmosphere of 5% CO 2 in air, thereafter the cells were incubated for 6 h under an atmosphere of 5% CO 2 and 1% O 2 (hypoxia) or for 6 h under an atmosphere of 5% CO 2 in air (normoxia). The medium was removed and washed with fresh medium. Confocal Fluorescence images were then obtained on Olympus IX83-FV3000.