A Bioorthogonal Antidote Against the Photosensitivity after Photodynamic Therapy

Abstract As an effective and non‐invasive treatment modality for cancer, photodynamic therapy (PDT) has attracted considerable interest. With the recent advances in the photosensitizing agents, the fiber‐optic systems, and other aspects, its application is extended to a wide range of superficial and localized cancers. However, for the few clinically used photosensitizers, most of them suffer from the drawback of causing prolonged photosensitivity after the treatment. As a result, post‐PDT management is also a crucial issue. Herein, a facile bioorthogonal approach is reported that can effectively suppress this common side effect of PDT in nude mice. It involves the use of an antidote that contains a black‐hole quencher BHQ‐3 conjugated with a bicyclo[6.1.0]non‐4‐yne (BCN) moiety and a tetrazine‐substituted boron dipyrromethene‐based photosensitizer. By using tumor‐bearing nude mice as an animal model, it is demonstrated that after PDT with this photosensitizer, the administration of the antidote can effectively quench the photodynamic activity of the residual photosensitizer by bringing the BHQ‐3 quencher close to the photosensitizing unit through a rapid click reaction. It results in substantial reduction in skin damage upon light irradiation. The overall results demonstrate that this simple and facile strategy can provide an effective means for minimizing the photosensitivity after PDT.


Introduction
Photodynamic therapy (PDT) has emerged as a promising treatment modality for cancer. [1]It involves the light irradiation of a photosensitizer localized in malignant tumor, followed by the interaction with the endogenous oxygen to generate cytotoxic reactive oxygen species (ROS), such as singlet oxygen.Apart from the direct killing of cancer cells through necrosis, apoptosis, and/or other forms of regulated cell death, the ROS generated can also damage the tumor vasculature, indirectly eradicating the tumor DOI: 10.1002/advs.202306207 by disrupting the oxygen and nutrient supply. [2]As the three components are individually non-toxic, the treatment exhibits minimal invasiveness and less systemic toxicity compared with the traditional anticancer therapies.The photodynamic action could be confined to the target site through precise delivery of the light, endowing a high spatiotemporal selectivity to this modality.By using tailor-made photosensitizing materials and advanced light technologies, it could also overcome the light penetration barrier. [3]Owing to these advantages, PDT has received growing attention as an innovative and effective procedure for cancer treatment, and there have been extensive studies to address the various challenges of PDT in recent years with a view to promoting its clinical applications. [4]ince the first PDT drug Photofrin (or porfimer sodium) being approved in Canada in 1993, [1c] considerable efforts have been devoted to developing more potent and safer photosensitizers with fewer side effects. [5]1d] For most of these clinically used photosensitizing agents, they suffer from the low tumor selectivity and slow metabolism.As a result, they tend to accumulate in the skin after systemic administration, causing the horrendous effect of prolonged photosensitivity. [6]Taking Photofrin as an example, the residual drug is present in all parts of the skin, which inevitably causes cutaneous toxicity upon exposure to sunlight. [7]According to the guidelines of U.S. Food and Drug Administration, all patients receiving this drug must avoid exposure of skin and eyes to direct sunlight or bright indoor light for at least 30 days (up to 90 days or more for some patients) to allow complete clearance of the drug from the body. [8]Hence, the posttreatment phototoxicity of photosensitizers remains a challenge that limits the clinical use of this modality.
With the goal of circumventing this problem, various strategies have been explored in laboratories.Apart from the approach of using tumor-targeting ligands to promote the active uptake of photosensitizers by cancer cells, [9] a wide range of activatable photosensitizers have also been developed, which ideally can be activated only by the stimuli in the tumor microenvironment, making them non-toxic at the non-target sites even upon light irradiation. [10]However, the actual outcome of employing these "smart" photosensitizers is affected by their intrinsic quenching efficiency, the extent of activation, the relevance of the stimuli as tumor biomarkers, as well as the specificity of the activation.As the difference in levels of most of the stimuli in cancer cells and normal cells is somewhat subtle, non-specific activation is usually unavoidable.
Recently, the concept of self-degradable photosensitizers has been proposed to address the post-PDT safety problem.Generally, these photosensitizers lose their photoactivities through degradation by the ROS generated or those present intrinsically in the cells (e.g., ClO -).A series of these photosensitizers based on the host-guest complexes of a boron dipyrromethene (BODIPY) and cucurbit [7]uril [11] or pillar [5]arene, [12] methylene blue analogues, [13] anthracene-bridged donor-acceptor chromophores, [14] or aggregation-induced emission dyes [15] have been reported.As light is generally required for the degradation of these photosensitizers, it is expected that the residual photosensitizers in the body cannot be degraded in the dark and can still cause photosensitivity upon exposure to light despite the effect may be attenuated along with the photodegradation.Therefore, it is of utmost importance to develop effective strategies for deactivation of photosensitizers after the photodynamic treatment.
To this end, Li et al. have recently developed a family of supramolecular organic frameworks [16] and naphthaleneincorporated tetracationic cyclophanes [17] that can adsorb some clinically used photosensitizers, including Photofrin, HiPorfin, Talaporfin, and Chlorin e6 in the body of mice through host-guest interactions.It has been found that the post-treatment of these hosts can suppress the sunlight-induced skin phototoxicity without affecting the PDT efficacy.We report herein an alternative strategy for deactivation of the residual photosensitizer through covalent bioorthogonal coupling with an antidote containing an effective quencher.Bioorthogonal chemistry involves chemical reactions that can take place specifically and efficiently under physiological conditions without interfering with the native biochemical processes. [18]By using a range of sophisticated click reactions, such as the copper-catalyzed alkyne-azide cycloaddition (CuAAC), Staudinger ligation, strain-promoted alkyne-azide cycloaddition (SPAAC), and inverse electron-demand Diels-Alder reaction (IEDDA), biomolecules can be studied in their native environments and manipulated for various applications, including protein and glycan imaging, identification of active enzymes, targeted drug delivery, and high-throughput screening of molecules against proteins. [19]19b,20] However, to the best of our knowledge, bioorthogonal chemistry has rarely been reported for deactivation of the residual drugs to reduce their side effects, including for post-PDT management. [21]

Working Principle
Figure 1 illustrates the concept of this bioorthogonal deactivating approach.It involves the use of a photosensitizer substituted with a tetrazine unit (PS-Tz) and a bicyclo[6.1.0]non-4-yne(BCN) conjugated with a quencher (BCN-Q).After PDT, the residual photosensitizer in the body, particularly on the skin, inevitably generates ROS upon exposure to light causing photosensitivity.By injecting the BCN-modified quencher, it can "click" with the residual tetrazine-substituted photosensitizer through the highly efficient IEDDA reaction between the two components. [22]The proximal quenching unit then effectively inhibits the photodynamic activity of the photosensitizer, thereby preventing the photosensitivity after the treatment.Thus, the BCN-modified quencher BCN-Q can function as a bioorthogonal antidote against this common side effect of PDT.As the photosensitizing unit is covalently linked to the quencher, the resulting conjugate is expected to be more robust than the non-covalent counterpart in the biological systems, which might undergo displacement and subsequently reactivation of the photosensitizer.

Molecular Design and Synthesis
Owing to the tunable spectroscopic and photophysical properties, as well as the ease of chemical modification and high stability, BODIPYs are highly versatile photosensitizers for PDT. [23]Therefore, a BODIPY dye with an extended conjugation was selected as the photosensitizer for this study, enabling it responsive in the far-red region.This compound, labeled as PS-Tz (Figure 1), was prepared in 38% yield by Knoevenagel condensation of our previously reported tetrazine-substituted BODIPY 1 [24] and the corresponding triethylene glycol monomethyl ether-substituted benzaldehyde 2 [25] (Scheme S1, Supporting Information) and characterized with NMR spectroscopy and electrospray ionization (ESI) mass spectrometry (Figures S1,S2, Supporting Information).The tetrazine unit was introduced to facilitate the coupling with the BCN-substituted quencher via the IEDDA reaction. [22]It is worth mentioning that while a tetrazine moiety can effectively quench the photoactivities of non--extended BODIPYs, [24,26] it is not a good quencher for distyryl BODIPYs. [27]It has been reported that tetrazines cannot effectively quench the fluorescence of dyes that emit in the far-red and near-infrared region. [28]or the antidote BCN-Q (Figure 1), it contains a BCN moiety for bioorthogonal coupling and a black-hole quencher BHQ-3 moiety.With a polyaromatic azo skeleton, this non-emissive azo dye is a well-known dark quencher for red-and far-red-emitting fluorophores through the Förster resonance energy transfer (FRET) mechanism owing to the appropriate spectral overlap, involving photochemical isomerization of the azo bridge to provide an effective non-emissive relaxation pathway. [29]Apart from the fluorescence emission, the ROS generation ability of a range of photosensitizers can also be effectively quenched by this dye via FRET. [30]30d] Its stability in phosphate-buffered saline (PBS) and the cell culture medium Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) was examined using high-performance liquid chromatography (HPLC).In both media, the chromatogram was virtually unchanged over a period of 14 days (Figure S3, Supporting Information), indicating that this conjugate possesses high stability in these biological media.

Bioorthogonal Quenching in Solution
The bioorthogonal quenching of PS-Tz by BCN-Q was first examined by monitoring the change in fluorescence spectrum of PS-Tz (1 μm) in PBS in the presence of 0.1% Tween 80 (v/v) with or without the presence of different concentrations of BCN-Q (1, 2, and 3 μm) over a period of 24 min.The surfactant Tween 80 was added to increase the solubility and reduce the aggregation of the compounds in this aqueous medium.It was found that in the absence of BCN-Q, the spectrum was virtually unchanged over this period of time (Figure S4a, Supporting Information).Upon addition of BCN-Q (1, 2, and 3 μm), the fluorescence band at ca. 700 nm was diminished largely and almost spontaneously (Figure S4b-d, Supporting Information).As shown in Figure 2a, which depicts the time-dependent variation of the fluorescence intensity at 702 nm, when 1 μm of BCN-Q was added, the intensity was reduced to the minimum after ca. 12 min, while the time was shortened to ca. 6 min when 2 or 3 μm of BCN-Q was used.The effective quenching could be attributed to the covalent linkage to the BHQ-3 moiety via an IEDDA reaction.To provide further evidence, the commercially available non-BCN-substituted BHQ-3 amine NH 2 -Q and a non-BHQ-3-conjugated BCN derivative 3 [31] were also used for comparison.It was found that in the presence of 3 μm of NH 2 -Q, the fluorescence intensity of PS-Tz (1 μm) was just slightly reduced over the whole period of time (Figure S4e, Supporting Information, and Figure 2a), showing that the click process was essential for effective quenching of the fluorescence of PS-Tz.As expected, without the quenching component, BCN 3 could not significantly change the fluorescence intensity of PS-Tz (Figure S4f, Supporting Information).
To demonstrate the generality of this approach and for comparison, we also used our previously reported azide-modified distyryl BODIPY, [32] labeled as PS-N 3 , instead of PS-Tz as the photosensitizer.With a reactive azide group, this compound was expected to couple with BCN-Q efficiently via the SPAAC reaction. [33]Therefore, it could also be used to demonstrate the bioorthogonal deactivation.Similarly, we monitored the change in fluorescence spectrum of this compound (1 μm) with or without the presence of BCN-Q (1, 2, and 3 μm) over a period of 24 min.As expected, its fluorescence at ca. 720 nm was also largely reduced in the presence of BCN-Q, and the rate of decrease was slightly faster when a higher concentration of BCN-Q was used (Figure S5, Supporting Information).As shown in Figure S5e (Supporting Information), more than 15 min was required to attain the lowest intensity, showing that the rate of quenching for PS-N 3 was slightly slower than that for PS-Tz (Figure 2a), which could be attributed to the less efficient SPAAC reactions compared with the IEDDA reactions. [34]Owing to the more efficient quenching by BCN-Q, PS-Tz was used for all the subsequent studies.
As the quenching of the fluorescence could be attributed to the formation of the clicked product, the conjugate of PS-Tz and BCN-Q was prepared for comparison.As shown in Scheme S2 (Supporting Information), the clicked product labeled as PS-Q could be prepared readily (in 95% yield) by mixing the two components in CH 3 CN, followed by purification by reserve-phase HPLC (Figure S6, Supporting Information) and characterization by ESI mass spectrometry (Figure S7, Supporting Information).As expected, the fluorescence of this conjugate in the aforementioned aqueous medium was virtually vanished as shown in Figure 2b, which also includes the fluorescence spectra of PS-Tz (1 μm) with or without the presence of different concentrations of BCN-Q (1, 2, and 3 μm) or NH 2 -Q (3 μm) recorded at 6 min after the mixing when the coupling was essentially completed.It can be seen that when 2 or 3 μm of BCN-Q was used, the resulting spectra did not show noticeable fluorescence as in the case of the conjugate PS-Q.Hence, it can be concluded that 2 equiv. of BCN-Q is already sufficient to fully quench the fluorescence of PS-Tz within 6 min, and this amount was used in the biological studies.
To quantitatively determine the quenching efficiency, the fluorescence quantum yields (Φ f ) of PS-Tz and PS-Q were measured and compared both in N,N-dimethylformamide (DMF) and in water with 0.1% Tween 80 (v/v).As shown in Figure S8a (Supporting Information), while a strong fluorescence band was observed at 707 nm for PS-Tz, the fluorescence of PS-Q was negligible as in the case of the quenching component BCN-Q in DMF.30d] The fluorescence quantum yield of PS-Tz in DMF was very close to those of our previously reported non-tetrazine-substituted dibromo distyryl BODIPYs, [35] which further supported that the tetrazine unit does not exert a significant quenching effect on the BODIPY core.The results in the aqueous medium were very similar with the value of Φ f decreasing from 0.31 for PS-Tz to 0.009 for PS-Q (Table 1).
Apart from the study of fluorescence quenching, we also investigated the effect of singlet oxygen formation upon the bioorthogonal coupling.As photosensitivity is mainly caused by ROS, the deactivation of photosensitizers in this aspect is of vital importance.Using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen probe, [36] the singlet oxygen generation efficiency of PS-Tz (1 μm) was monitored before and after the treatment with different concentrations of BCN-Q (1, 2, and 3 μm) in the same aqueous medium for 6 min.As shown in Figure 2c, the absorbance of the DPBF's absorption at 417 nm decreased rapidly upon light irradiation ( > 610 nm) for PS-Tz, indicating that it is an efficient photosensitizer.However, after the treatment with BCN-Q for 6 min, the singlet oxygen generation ability of PS-Tz was largely suppressed.The efficiency was comparable with that of the covalent conjugate PS-Q, particularly when 2 or 3 equiv.of BCN-Q was used.
Similarly, the quenching efficiency in this aspect was quantitatively evaluated by comparing the singlet oxygen quantum yields (Φ Δ ) of PS-Tz and PS-Q in DMF and in water with 0.1% Tween 80 (v/v).For the measurements in DMF, DPBF was again used as the singlet oxygen scavenger [36] and ZnPc was used as the reference (Φ Δ = 0.56), [30d] while in the aqueous medium, 9,10anthrancenediyl-bis(methylene)dimalonic acid (ABDA) was used as the probe [36] with reference to methylene blue (MB) (Φ Δ = 0.52 in water). [37]As shown in Figure S9 (Supporting Information),  d) In the presence of 0.1% Tween 80 (v/v); e) Relative to MB (Φ Δ = 0.52 in water) using ABDA as the singlet oxygen scavenger; f) The value was too weak to be measured.
PS-Tz could effectively consume both singlet oxygen scavengers upon irradiation, though its efficiency was lower than that of the reference compounds, from which the Φ Δ values were determined to be 0.12 in DMF and 0.38 in the aqueous medium.
In contrast, the singlet oxygen generation efficiency of PS-Q was negligible.The Φ Δ values were decreased to 0.002 and 0.02 in these two media, respectively (Table 1).
As mentioned above, it is believed that the quenching of these photoactivities is mainly through the FRET mechanism.It is based on the substantial spectral overlap between the fluorescence spectrum of PS-Tz and the electronic absorption spectrum of BCN-Q (Figure S8b, Supporting Information) as well as the highly efficient IEDDA reaction that connects the two components almost spontaneously and brings them in close proximity.The latter is expected to be a more important factor governing the quenching efficiency by FRET as reported previously. [38]To reveal the possibility of quenching by ground-state complex formation, the electronic absorption spectra of PS-Tz, BCN-Q, and PS-Q in DMF were compared (Figure S8c, Supporting Information).It was found that the spectrum of PS-Q is essentially a superposition of the sum of the spectra of PS-Tz and BCN-Q.30a]

In Vitro Bioorthogonal Deactivation
The bioorthogonal deactivation of PS-Tz by BCN-Q was further demonstrated at the cellular level.HT29 human colorectal adenocarcinoma cells were first incubated with PS-Tz (1 μm) for 6 h and then in the culture medium with or without NH 2 -Q or BCN-Q (2 μm) for 1 h.For comparison, the cells were also incubated with PS-Q (1 μm) for 6 h without the post-treatment or simply with BCN-Q (2 μm) for 1 h.The corresponding confocal images and quantified intracellular fluorescence intensities are shown in Figure 3a,b, respectively.Bright fluorescence was observed for the cells being treated with PS-Tz, and the fluorescence intensity remained very strong when the cells were post-treated with NH 2 -Q.In contrast, for the cells being post-treated with BCN-Q, the fluorescence intensity was reduced by ca.5-fold, which was comparable with that for the cells being incubated with PS-Q, indicating that PS-Tz was essentially fully deactivated by BCN-Q inside the cells.As expected, the intracellular fluorescence of the dark quencher BCN-Q was negligible.
A similar study was performed using two other cell lines, namely A549 human lung carcinoma cells and HepG2 human hepatocarcinoma cells.As shown in Figure 3c,d, the intracellular fluorescence intensity of PS-Tz was also largely reduced when the cells were post-treated with BCN-Q, and the extent (ca.5-fold) was similar to that for HT29 cells.The results showed that BCN-Q could effectively deactivate the fluorescence emission of PS-Tz inside a range of cancer cells through bioorthogonal conjugation.
The study was then extended to examine the bioorthogonal deactivation in ROS formation inside the cells, using 2′,7′dichlorodihydrofluorescein diacetate (H 2 DCFDA) as the ROS probe. [39]Upon internalization, this probe would be deacetylated by the intracellular esterase and subsequently oxidized by the intracellular ROS to form the highly fluorescent 2′,7′-dichlorofluorescein (DCF), which can be detected readily by confocal microscopy.In this study, the cells were incubated with PS-Tz (1 μm) for 6 h and then in the culture medium with or without NH 2 -Q or BCN-Q (2 μm) for 1 h or incubated simply with PS-Q (1 μm) for 6 h, followed by the incubation with H 2 DCFDA (50 μm) for 30 min.The cells were then either left in the dark or irradiated with red light ( > 610 nm, 23 mW cm −2 ) for 20 min.Figure 4a shows the confocal fluorescence images of the cells under all these conditions.It can be seen that in the absence of light irradiation, the fluorescence was negligible for all the cells.Upon light irradiation, bright green fluorescence of DCF could be observed for the cells being incubated with PS-Tz and those with post-treatment with NH 2 -Q.For the cells with posttreatment with BCN-Q, the fluorescence of DCF was significantly reduced, and the intensity was comparable with that by using the conjugate PS-Q for incubation.The attenuation effect was also observed for A549 and HepG2 cells (Figure 4b), showing that BCN-Q could also effectively deactivate the ROS generation ability of PS-Tz inside the cells.
Since PS-Tz could effectively generate ROS inside the cells upon light irradiation, it was expected to exhibit high photocytotoxicity.To examine this property, all the three cell lines were incubated with different concentrations (up to 8 μm) of PS-Tz for 6 h, followed by the dark or light ( > 610 nm, 23 mW cm −2 ) treatment for 20 min.As shown in Figure 4c-e, while the photosensitizer was noncytotoxic in the dark, it exhibited high photocytotoxicity.The half-maximal inhibitory concentration (IC 50 value) was determined to be 2.0, 3.6, and 4.4 μm for HT29, A549, and HepG2 cells, respectively.Upon post-incubation of the cells with 2 equiv. of BCN-Q for 1 h, the cells remained viable in the absence of light.Even upon light irradiation, the cell viability was only decreased by at most 20% at a drug dose of 8 μm, showing that the photocytotoxicity of PS-Tz was greatly reduced upon posttreatment with BCN-Q.These results were consistent with those of the study of intracellular ROS generation as reported above and further demonstrated that the photodynamic activity of PS-Tz could be effectively inhibited by this bioorthogonal antidote.
To reveal the toxicity of the resulting conjugate, the dark and photocytotoxicity of PS-Q was examined against all the three cell lines.As shown in Figure S10a-c (Supporting Information), its cytotoxicity was negligible under all the conditions.For the antidote BCN-Q, its cytotoxicity was also not noticeable (Figure S10d, Supporting Information).These results showed that both the clicked product and the antidote were not cytotoxic.To provide further evidence, hemolysis assay was also performed for the photosensitizer PS-Tz, the antidote BCN-Q, and the conjugate PS-Q using rabbit red blood cells.The cells were incubated with different concentrations of these compounds at 37 °C for 6 h, and then the amount of hemoglobin released was determined spectroscopically.It was found that even at a dose of 40 μm, cell lysis was not noticeable for all these compounds (Figure S11, Supporting Information), showing that they exhibited high blood compatibility.

In Vivo Bioorthogonal Deactivation
With these encouraging in vitro results, we further examined the bioorthogonal deactivation effect using a mouse model.Female Figure 5a shows the near-infrared fluorescence images of the mice recorded with an infrared imaging system (excitation wavelength = 680 nm, emission wavelength ≥ 700 nm) at 24 h postinjection of PS-Tz and after the intravenous injection of BCN-Q or PBS over a period of further 24 h.Strong fluorescence was clearly observed throughout the whole body of the mice at 24 h postinjection of PS-Tz.After further injection with BCN-Q, the fluorescence intensity of the whole body was significantly reduced along with time compared with the control with post-injection with PBS.Even just after 10 min, the effect was remarkable, which could be a result of the fast IEDDA reaction between PS-Tz and BCN-Q.This feature is highly desirable for antidotes.
To quantitatively evaluate the deactivation efficiency, the fluorescence intensity per unit area of the skin at the backside was determined at different time points over a period of 24 h after the injection of BCN-Q or PBS at 24 h post-injection of PS-Tz.As shown in Figure 5b, while the fluorescence intensity was slightly increased with time upon intravenous injection of PBS, the intensity was rapidly and significantly decreased when BCN-Q was subsequently injected.At 24 h post-administration of BCN-Q, the mice were sacrificed.The skin tissues from different positions, namely the leg, back, and belly, as well as some major organs, including the spleen, lung, liver, kidney, and heart were then harvested and imaged.As shown in Figure 5c-f, the fluorescence intensities of all the skin tissues and organs of the BCN-Q-treated mice were significantly weaker than those of the PBS-treated control, which were in accordance with the whole-body imaging results.For the skin tissues, the reduction in fluorescence intensity was in the range of 55-72%.These results clearly demonstrated that BCN-Q could effectively deactivate PS-Tz in vivo, reducing its fluorescence in the skin and major organs.
The in vivo deactivation via bioorthogonal coupling was further demonstrated using liquid chromatography -mass spectrometry (LC-MS).After the intravenous injection of PS-Tz and BCN-Q as mentioned above, blood samples were collected from the mice at 10 min and 8 h after the injection of BCN-Q, which were then subject to LC-MS analysis.Figure S12a (Supporting Information) shows the HPLC chromatogram of a blood sample collected at 10 min post-injection.Apart from the signals due to PS-Tz and BCN-Q, an intense signal with a retention time of 28.7 min was also observed.Its ESI mass spectrum showed the base peak at m/z 872 and another signal at m/z 1742, which could be assigned to the [M+H] 2+ and [M] + ions of the clicked product PS-Q, respectively (Figure S12b, Supporting Information).For the sample collected at 8 h post-injection of BCN-Q, the presence of PS-Q was also confirmed by LC-MS analysis (Figure S12c,d, Supporting Information).In the HPLC chromatogram, the signal due to PS-Tz was almost invisible, showing that the click reaction had already been completed in the blood within 8 h.

In Vivo PDT Followed by Bioorthogonal Deactivation
Before the study of in vivo PDT using PS-Tz, its biodistribution in HT29 tumor-bearing nude mice after intravenous injection (20 nmol, 100 μL) was first monitored over a period of 48 h  (c,d) the skin tissues and (e,f) some major organs harvested from the above two groups of mice at 24 h postinjection of BCN-Q or PBS.Skin 1, 2, and 3 are the skin tissues taken from the leg, back, and belly, respectively.Data are reported as the mean ± standard deviation (SD) of three mice in each group.
using the aforementioned infrared imaging system.As shown in Figure S13a (Supporting Information), the fluorescence intensity in the body of the mice grew gradually along with time.At the tumor site, the fluorescence intensity was also increased with time and almost reached the maximum after 24 h (Figure S13b, Supporting Information).Ex vivo study at 48 h post-injection showed that the fluorescence intensity per unit area in the tumor was comparable with, if not higher than, that in some major organs (Figure S13c,d, Supporting Information).These results showed that PS-Tz can accumulate in tumor along with time, which enables it to be used for photodynamic elimination of tumor.
The PDT efficacy of PS-Tz in HT29 tumor-bearing nude mice and the bioorthogonal deactivation effect of BCN-Q after the photodynamic treatment were then investigated according to the procedure outlined in Figure 6a.In short, HT29 cells were inoculated subcutaneously on the back of the mice.When the tumor volume reached a size of ≈60 mm 3 , the mice were injected intravenously with PS-Tz (20 nmol).After 24 h when the accumulation of this compound in the tumor almost reached the maximum as shown in Figure S13b (Supporting Information), the tumor was irradiated with a diode laser at 675 nm operated at 0.3 W for 10 min (total fluence = 180 J cm −2 ) (labeled as Laser-1) to initiate the photodynamic treatment.The mice were then injected intravenously with BCN-Q (40 nmol).After further 24 h at which virtually all the PS-Tz had been quenched by BCN-Q as shown in Figure 5b, the backside of the mice was irradiated with another laser (675 nm, 0.6 W, 360 J cm −2 , labeled as Laser-2) to study the bioorthogonal deactivation effect of BCN-Q.As the fluorescence intensity per unit area of the tumor was higher than that of the skin as shown in Figure S13a (Supporting Information), we used a stronger light dose arbitrary set as 2-fold to ensure that its effect on the skin is substantial and should be at least as strong as that on the tumor.The mice were monitored for a period of 14 days after the injection of BCN-Q, and then they were sacrificed for detailed analysis using various controls.
We first focused on the in vivo PDT efficacy of PS-Tz. Figure 6b shows the tumor growth curves for the treatment group mentioned above and various controls.It can be seen that for the negative control of intravenous injection of PBS without applying Laser-1 at the tumor, the tumor grew continuously over 14 days.A similar situation was observed when the mice were just injected with PS-Tz without the laser treatment.In contrast, when the mice were treated with PS-Tz followed by the treatment of Laser-1, the growth of the tumor was greatly inhibited, regardless of whether BCN-Q and Laser-2 were applied, showing that PS-Tz exhibited a strong antitumor PDT effect, and the post-treatment of BCN-Q and Laser-2 did not affect its PDT efficacy.Figure S14 (Supporting Information) shows the representative images of the mice before and after the different treatments.For the groups receiving the treatment of Laser-1, a scar could clearly be seen at the tumor which faded out gradually.It should be caused by the photodynamic effect of PS-Tz instead of the laser irradiation as we confirmed previously [30d] and below.
On day 14 after the various treatments, the mice were sacrificed and the tumors were harvested.Both the weight and the size of the tumors were significantly higher/larger for the PBS and PS-Tz control groups compared with the other two groups receiving the treatment of PS-Tz and Laser-1 (Figure 6c,d).These results were fully consistent with the tumor growth curves shown in Figure 6b.The tumor slides were then subjected to hematoxylin and eosin (H&E) staining for histological analysis.As shown in Figure 6e (upper row), while no abnormality was observed for the PBS and PS-Tz control groups, notable necrotic and apoptotic cells were detected in the slides of the tumors being treated with PS-Tz and Laser-1, reflecting a high degree of photodamage.These tumor slides were further examined using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Figure 6e, lower row).Bright green fluorescence was observed only in the tumor tissues from the mice receiving the photodynamic treatment of PS-Tz, which indicated the occurrence of extensive cell apoptosis.For the PBS and PS-Tz without the treatment of Laser-1 control groups, the fluorescence was not noticeable, showing that the tumor remained essentially intact.
To examine the general toxicity of these treatments, we also harvested the heart, kidneys, liver, lung, and spleen of the mice on Day 14 and performed H&E staining.It was found that there was no toxicological lesion or major abnormality in the stained organ slides (Figure S15, Supporting Information), showing that all these treatments did not induce significant toxicity to these internal organs.We also monitored the body weight of the mice for each treatment group over a period of 14 days (Figure 6f).The negligible change also suggested that the treatments did not cause notable adverse effects to the mice.
As photosensitivity is a very common side effect of PDT, the deactivating effect of the antidote BCN-Q was focused on the skin tissues.We first monitored the mice of groups (1), (3), and (4) as described above, for which Laser-2 was applied, over a period of 14 days from the administration of BCN-Q. Figure 7a shows the photographs of one of the mice in each group, while those of the other three mice in each group are given in Figures S16-S18 (Supporting Information).It can be seen that the results were highly consistent.For the PBS-treated mice (i.e., group 1), no noticeable photosensitivity was observed at the back of the mice, confirming that this power of laser irradiation would not cause significant damage to the skin.In contrast, the mice being sequentially treated with PS-Tz, Laser-1, and Laser-2 (i.e., group 3) suffered from severe skin erythema and edema at the irradiated site of Laser-2.The condition was not significantly improved over 14 days.However, when BCN-Q was administered after the PDT treatment (i.e., group 4), only slight erythema was observed at the irradiated site, which recovered in a few days.
The different extent of skin damage was further validated using H&E and TUNEL staining of the corresponding skin tissues.For the PBS-treated mice, the H&E-stained skin tissues remained connected and did not show obvious pathological changes (Figure 7b).A similar result was observed in the skin tissues from the mice receiving post-treatment of BCN-Q after the photodynamic treatment of PS-Tz.Without the post-treatment, apoptotic keratinocytes (as indicated by black arrows) and vacuoles could clearly be seen in the tissues.The former is smaller than the healthy keratinocytes and is characterized by the dark and condensed nucleic chromatin.For the TUNEL-stained slides (Figure 7c), damaged epidermis and hair follicles were only observed in the bright field images (as indicated by black arrows) for the mice of group 3 accompanied with strong green fluorescence appeared after the TUNEL staining, while minimal skin damage and green signal were observed for the control groups 1 and 4. All these results demonstrated that the photodynamic effect of PS-Tz caused severe damage to the skin tissues, and BCN-Q could largely reduce the photosensitivity induced by this photosensitizer in mice.

Conclusion
In summary, we have developed a novel and unprecedented bioorthogonal strategy for addressing the problem of photosensitivity induced after PDT.It involves the use of the antidote BCN-Q, which contains a BCN moiety that can rapidly click with the tetrazine substituent of the photosensitizer PS-Tz and a BHQ-3 quencher that can effectively quench the fluorescence emission and ROS generation of PS-Tz after the covalent linkage through a FRET process.The efficient quenching of these photoactivities has been demonstrated in PBS and a range of cancer cell lines using various controls.Using nude mice as an animal model, it has also been shown that the injection of this antidote can effectively deactivate the residual photosensitizer in the body of the mice without affecting the PDT efficacy.In particular, the skin tissues showed minimal damage after the post-treatment with BCN-Q after the PDT with PS-Tz as shown by the physical appearance of the skin as well as the H&E and TUNEL staining.Compared with the approach of using activatable photosensitizers to enhance the tumor specificity and reduce the unwanted photodamage to nontarget sites, this highly efficient bioorthogonal deactivating strategy is not affected by the subtle difference in the levels of the corresponding stimuli in cancer and normal cells.Its straightforward "click-and-quench" mechanism ensures the residual photosensitizer in the body can be deactivated effectively after the photodynamic treatment.The overall results demonstrate that this bioorthogonal approach can effectively suppress the photosensitivity after PDT in nude mice and would be potentially useful in minimizing this common side effect of PDT, thereby promoting its clinical application.

Experimental Section
General: All reactions were performed under an atmosphere of nitrogen.DMF and CH 2 Cl 2 were dried using an INERT solvent purification system.PBS at pH 7.4 was used in all the studies.All other solvents and reagents were used as received.Chromatographic purification was performed on silica gel (Macherey-Nagel 230-400 mesh) with the indicated eluent.Compounds 1, [24] 2, [25] PS-N 3 , [32] and BCN-Q [30d] were prepared according to the literature procedure.
Instrumentation: 1 H and 13 C{ 1 H} NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer or a Bruker AVANCE III 500 MHz spectrometer in CDCl 3 .Spectra were referenced internally by using the residual solvent ( 1 H:  7.26) or solvent ( 13 C:  77.2) resonance relative to SiMe 4 .ESI mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer.Electronic absorption and steady-state fluorescence spectra were taken on a Cary 5G UV-Vis-NIR spectrophotometer and a HORIBA FluoroMax-4 spectrofluorometer, respectively.Reversephase HPLC separation was performed on an Apollo-C18 column (5 μm, 4.6 mm × 150 mm) at a flow rate of 1 mL min −1 for analytical purpose or on a XBridge BEH300 Prep C18 column (5 μm, 10 mm × 250 mm) at a flow rate of 3 mL min −1 for preparative purpose, using a Waters system equipped with a Waters 1525 binary pump and a Waters 2998 photodiode array detector.The solvents used for HPLC analysis were of HPLC grade.LC-MS studies were performed on a XSelect CSH C18 column (5 μm, 4.6 mm × 250 mm) at a flow rate of 0.8 mL min −1 using a Waters system equipped with a Waters Quaternary Solvent Manager-R, a Waters 2998 photodiode array detector, a Waters 2475 fluorescence detector, and a Waters single quadrupole detector 2. The solvents used were of LC-MS grade.
Preparation of PS-Tz: BODIPY 1 (120 mg, 0.21 mmol) and benzaldehyde 2 (224 mg, 0.84 mmol) were dissolved in toluene (50 mL).Acetic acid (0.3 mL) and piperidine (0.3 mL) were then added.The mixture was heated under reflux with a Dean-Stark trap.After the consumption of 1 as indicated by thin-layer chromatography, the mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure.Water (150 mL) was then added to the residue, and the crude product was extracted with CH 2 Cl 2 (100 mL × 3).The combined organic phase was dried over anhydrous Na 2 SO 4 , and the solvent was evaporated under reduced pressure.The crude product was purified by column chromatography on silica gel with CH 2 Cl 2 /CH 3 OH (70:1 v/v) as eluent to afford PS-Tz as a green solid (85 mg, 38%).Bioorthogonal Quenching in Solution: The deactivation of PS-Tz and PS-N 3 by BCN-Q was performed in a 1 cm × 1 cm quartz cuvette.Stock solutions of PS-Tz and PS-N 3 were prepared in DMF (2 mm), which were diluted to 1 μm with PBS in the presence of 0.1% Tween 80 (v/v).Another stock solution of BCN-Q in DMF (2 mm) was also prepared, which was then added to the above solution of PS-Tz or PS-N 3 to attain the final concentration of BCN-Q to 1, 2, and 3 μm, respectively.The fluorescence spectra were recorded in the range of 650-800 nm at different time points upon excitation at 610 nm.To study the bioorthogonal deactivation in ROS formation, a mixture of DPBF (30 μm) and PS-Q or PS-Tz (1 μm) with or without the treatment with BCN-Q (1, 2, and 3 μm) for 6 min in PBS in the presence of 0.1% Tween 80 (v/v) was irradiated with red light from a 300 W halogen lamp after passing through a water tank for cooling and a color filter with a cut-on wavelength at 610 nm (Newport).The absorption maximum of DPBF at 417 nm was monitored along with the irradiation time.For the measurements of fluorescence and singlet oxygen quantum yields, the procedures as described previously were followed. [40]ell Lines and Culture Conditions: HT29 cells (ATCC, no.HTB-38) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, no.23400-021).A549 (ATCC, no.CCL-185) and HepG2 (ATCC, no.HB-8065) cells were maintained in DMEM (ThermoFisher Scientific, no.12100-046).Both media were supplemented with FBS (10%) and a penicillin-streptomycin solution (100 unit mL −1 and 100 mg mL −1 , respectively).All the cells were grown at 37 °C in a humidified 5% CO 2 atmosphere.
Confocal Fluorescence Microscopic Study: Approximately 2 × 10 5 HT29, A549, and HepG2 cells in the corresponding culture medium (2 mL) were seeded on glass-bottom confocal dishes and incubated at 37 °C overnight in a humidified 5% CO 2 atmosphere.After removal of the medium, the cells were rinsed with PBS and incubated in the medium with PS-Tz (1 μm) for 6 h.After discarding the medium and rinsing the cells with PBS twice, the cells were further incubated in the medium with or without NH 2 -Q or BCN-Q (2 μm) for 1 h.For comparison, the cells were also incubated with the conjugate PS-Q (1 μm) instead of PS-Tz for 6 h without the posttreatment or simply with BCN-Q (2 μm) for 1 h.After being rinsed with PBS again, all the cells were replenished with Hank's Balanced Salt Solution (HBSS) (1 mL) before being examined using a Leica TCS SP8 high-speed confocal microscope equipped with a solid-state 638 nm laser.The photosensitizer was excited at 638 nm and its fluorescence was monitored at 650-750 nm.The images were digitized and analyzed using a Leica Application Suite X software.
Study of Intracellular ROS Generation: HT29, A549, and HepG2 cells, which had been treated as described above, were further stained with H 2 DCFDA (50 μm) for 30 min.The cells were then rinsed with PBS for three times followed by the dark or light treatment at ambient temperature for 20 min.The light source consisted of a 300 W halogen lamp, a water tank for cooling, and a colored glass filter with a cut-on wavelength at 610 nm (Newport).The fluence rate ( > 610 nm) was 23 mW cm −2 .Illumination of 20 min led to a total fluence of 28 J cm −2 .The fluorescence of DCF in these cells was imaged using confocal fluorescence microscopy.The DCF was excited at 488 nm, and the fluorescence was monitored at 515-580 nm.
Study of Photocytotoxicity: Approximately 1 × 10 4 HT29, A549, and HepG2 cells per well in the corresponding culture medium were inoculated in 96-well plates and incubated at 37 °C overnight in a humidified 5% CO 2 atmosphere.The stock solution of PS-Tz in DMF (2 mm) was diluted to various concentrations with the culture medium.The cells were then incubated with 100 μL of these drug solutions for 6 h.After being rinsed with PBS for three times, the cells were further incubated in the medium with or without two equiv. of BCN-Q for 1 h.After that, these cells were rinsed again with PBS and refed with 100 μL of the culture medium before being irradiated at ambient temperature with the above light source for 20 min (total fluence = 28 J cm −2 ).Cell viability was determined by means of a colorimetric MTT assay as described previously. [40]emolysis Assay: The blood compatibility of PS-Tz, BCN-Q, and PS-Q was evaluated with a hemolysis assay.Fresh rabbit blood was obtained from the Laboratory Animal Services Centre of The Chinese University of Hong Kong.A blood sample (2 mL) was centrifuged at 9000 rpm for 10 min to isolate the red blood cells (RBCs).The red pellet on the bottom was washed with PBS for three times.After that, these RBCs (400 μL) were mixed with PBS (9.6 mL) to give a RBC suspension (4% v/v).Stock solutions of PS-Tz, BCN-Q, and PS-Q in DMF (2 mM) were diluted with PBS to various concentrations (up to 40 μm).These solutions (50 μL) were then mixed with the RBC suspension (100 μL).After incubation at 37 °C in the dark for 6 h, the mixtures were centrifuged at 1500 rpm for 5 min.Aliquots (100 μL) of the supernatant were transferred to 96-well plates, and the hemoglobin released was monitored at 540 nm using a microplate reader (Tecan Spark 10 m).PBS and 0.5% Triton X-100 were used as the negative and positive controls, respectively.The percentage of hemolysis was calculated using the following equation: A 540 in solution − A 540 in PBS A 540 in Triton X − 100 − A 540 in PBS × 100 (1) In Vivo Bioorthogonal Deactivation: Female BALB/c nude mice (20-25 g) were obtained from the Laboratory Animal Services Centre of The Chinese University of Hong Kong.All animal experiments had been approved by the Animal Experimentation Ethics Committee of the University (Ref.No. 21-005-GRF).The mice were kept under a pathogen-free condition with free access to food and water.To evaluate the in vivo quenching efficiency of BCN-Q, the mice were intravenously injected with PS-Tz in PBS in the presence of 0.1% Tween 80 (v/v) (20 nmol, 100 μL).After being kept in the dark for 24 h, the mice were intravenously injected with BCN-Q in PBS (40 nmol, 200 μL) or PBS (200 μL).The fluorescence images of the mice were captured before and after the injection at different time points up to 24 h from the injection of BCN-Q or PBS, using an Odyssey infrared imaging system (excitation wavelength = 680 nm, emission wavelength ≥ 700 nm).The images were digitized and analyzed using an Odyssey imaging system software (9201-500).After the in vivo imaging study, the animals were euthanized at 24 h post-injection of BCN-Q or PBS.The skin tissues and major organs were harvested, and their fluorescence intensities were measured.Three mice were used for each group LC-MS Analysis of In Vivo Bioorthogonal Deactivation: After sequential intravenous injection of PS-Tz and BCN-Q as described above, blood sam-

Figure 1 .
Figure 1.Working principle of the bioorthogonal antidote BCN-Q for deactivation of the residual photosensitizer PS-Tz after the photodynamic treatment.

Figure 2 .
Figure 2. a) Change in the fluorescence intensity at 702 nm of PS-Tz (1 μm) with or without the presence of different concentrations of BCN-Q (1, 2, and 3 μm) or NH 2 -Q (3 μm) over a period of 24 min ( ex = 610 nm).b) Comparison of the fluorescence spectra of PS-Q (1 μm) and the above mixtures recorded at 6 min after the mixing ( ex = 610 nm).c) Comparison of the rates of decay of DPBF (initial concentration = 30 μm) sensitized by PS-Q (1 μm), PS-Tz (1 μm), and mixtures of PS-Tz (1 μm) and BCN-Q (1, 2, and 3 μm) after mixing for 6 min.The mixtures were irradiated ( > 610 nm) during the measurements.The solvent was PBS in the presence of 0.1% Tween 80 (v/v) for all the above measurements.

Figure 3 .
Figure 3. a) Bright field, fluorescence, and the merged confocal images of HT29 cells after incubation with PS-Tz (1 μm) for 6 h and then in the culture medium with or without NH 2 -Q or BCN-Q (2 μm) for 1 h or incubation simply with PS-Q (1 μm) for 6 h or BCN-Q (2 μm) for 1 h.b) Quantified fluorescence intensities for the cells being treated as described in (a).c) Bright field, fluorescence, and the merged confocal images of A549 and HepG2 cells after incubation with PS-Tz (1 μm) for 6 h and then in the culture medium with or without BCN-Q (2 μm) for 1 h.d) Quantified fluorescence intensities for the cells being treated as described in (c).For (b) and (d), data are reported as the mean ± standard error of the mean (SEM) of three independent experiments (n = 25 cells).

Figure 4 .
Figure 4. a) Intracellular ROS generation efficiency of PS-Tz (1 μm) with or without post-treatment with NH 2 -Q or BCN-Q (2 μm) for 1 h and of the conjugate PS-Q (1 μm), as reflected by the fluorescence intensity of DCF, in HT29 cells both in the absence and presence of light irradiation ( > 610 nm, 23 mW cm −2 ) for 20 min.b) Intracellular ROS generation efficiency of PS-Tz (1 μm) with or without post-treatment with BCN-Q (2 μm) for 1 h in A549 and HepG2 cells both in the absence and presence of light irradiation ( > 610 nm, 23 mW cm −2 ) for 20 min.Cytotoxicity of PS-Tz against c) HT29, d) A549, and e) HepG2 cells with or without post-treatment with BCN-Q (2 equiv.)for 1 h both in the absence and presence of light irradiation ( > 610 nm, 23 mW cm −2 , 28 J cm −2 ).Data are reported as the mean ± SEM of three independent experiments, each performed in quadruplicate.

Figure 5 .
Figure 5. a) Near-infrared ( ≥ 700 nm) fluorescence images of the nude mice at 24 h post-injection of PS-Tz in PBS in the presence of 0.1% Tween 80 (v/v) (20 nmol, 100 μL) and after intravenous injection of BCN-Q in PBS (40 nmol, 200 μL) or simply PBS (200 μL) over a period of further 24 h.b) Change in fluorescence intensity per unit area of the skin at the backside of the mice after the above treatments over a period of 24 h.Ex vivo images and quantified fluorescence intensities of(c,d) the skin tissues and (e,f) some major organs harvested from the above two groups of mice at 24 h postinjection of BCN-Q or PBS.Skin 1, 2, and 3 are the skin tissues taken from the leg, back, and belly, respectively.Data are reported as the mean ± standard deviation (SD) of three mice in each group.

Figure 6 .
Figure 6.a) Timeline for studying the PDT efficacy of PS-Tz in HT29 tumor-bearing nude mice and the bioorthogonal deactivation effect of BCN-Q after the photodynamic treatment.b) Tumor growth curves for HT29 tumor-bearing nude mice after different treatments: 1) intravenous injection with PBS with the treatment of Laser-2, 2) intravenous injection with PS-Tz without laser irradiation, 3) intravenous injection with PS-Tz followed by the treatment of Laser-1 and Laser-2, and 4) intravenous injection with PS-Tz with the treatment of Laser-1, followed by intravenous injection with BCN-Q with the treatment of Laser-2 (n = 4 for each group).Drug dose: 100 μL of PS-Tz (20 nmol) in PBS in the presence of 0.1% Tween 80 (v/v); 200 μL of BCN-Q (40 nmol) in PBS.Laser-1: 675 nm, 0.3 W, 180 J cm −2 ; Laser-2: 675 nm, 0.6 W, 360 J cm −2 .c) Weights and d) photographs of the tumors in the mice harvested on Day 14 after the above treatments.e) H&E and TUNEL-stained images of the tumor slices of the mice after the above treatments.Scale bar: 50 μm for the H&E-stained images.The apoptotic cells and the cell nucleus were stained by TUNEL (green) and Hoechst 33342 (blue), respectively.f) Variation of the body weight of the mice receiving the above treatments over a period of 14 days.For (b), (c), and (f), data are reported as the mean ± SD of four mice in each group.n.s., not significant; ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 7 .
Figure 7. a) Photographs of one of the mice of each of the groups 1, 3, and 4 as described above over a period of 14 days.The irradiated sites of Laser-2 are indicated with ovals.b) H&E-stained images of the skin tissues of the mice after the above treatments.Scale bar: 100 μm.c) TUNEL-stained images of the skin tissues of the mice after the above treatments.The apoptotic cells and the cell nucleus were stained by TUNEL (green) and Hoechst 33342 (blue), respectively.

Table 1 .
Fluorescence and singlet oxygen generation properties of PS-Tz and PS-Q in DMF and in water with 0.1% Tween 80 (v/v).