Nitroreductase-activatable theranostic molecules with high PDT

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Biological and Medical Applications of Materials and Interfaces

Nitroreductase-activatable theranostic molecules with high PDT efficiency under mild hypoxia based on a TADF fluorescein derivative Zhiwei Liu, Fengling Song, Wenbo Shi, Gagik Gurzadyan, Huiyi Yin, Bo Song, Ri Liang, and Xiaojun Peng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04488 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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ACS Applied Materials & Interfaces

Nitroreductase-Activatable Theranostic Molecules with High PDT Efficiency under Mild Hypoxia Based on a TADF Fluorescein Derivative Zhiwei Liu,† Fengling Song,†‡* Wenbo Shi,§ Gagik G. Gurzadyan, † Huiyi Yin,† Bo Song,§ Ri Liang,† Xiaojun Peng† †

State Key Laboratory of Fine Chemicals, §School of Chemistry, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P.R. China. ‡ Institute of Molecular Sciences and Engineering, Shandong University, Qingdao 266237, P. R. China.

KEYWORDS: Photodynamic therapy, Hypoxia, Cancer theranostics, Thermally activated delayed fluorescence, Nitroreductase, Photoinduced electron transfer ABSTRACT: High specificity detection and site-specific therapy are still the mainly challenges for theranostic anti-cancer prodrugs. In this work, we reported two smart activatable theranostic molecules based on a thermally activated delayed fluorescence (TADF) fluorescein derivative. Nitroreductase (NTR) induced by mild hypoxia microenvironment of solid tumor was used to activate the fluorescence and photodynamic therapy (PDT) efficiency by employing the intramolecular photoinduced electron transfer (PET) mechanism. A high PDT efficiency under 10% oxygen concentration was achieved, which is better than that of porphyrin (PpIX), a traditional photosensitizer. Such an excellent PDT efficiency can be attributed to lysosomes disruption, because the theranostic molecule can specifically enter the lysosomes of cells. Importantly, the strategy of targeting the mild hypoxic cells in the edge of tumor tissue could heal the “Achilles’ heel” of traditional PDT. We believe this theranostic molecule has a high potential to be applied in clinical investigation as a theranostic anti-cancer prodrug.

INTRODUCTION Theranostic anti-cancer prodrugs have caused extensive concern in the last decade, because they can diagnose and treat the nidus region at the same time.1-6 Traditional theranostic prodrugs are constituted by a fluorophore moiety for diagnosis response signal and a chemotherapy prodrug moiety for treatment. This combination exists some problems, such as complicated structure which is difficult to be synthesized, unpredictable effect of the prodrug structure when linked to the fluorophore, and the aporia of the drug site-specific release. Among them, the lack of the drug site-specific release will lead to irreversible damage to normal tissue which limits their practical application. Photodynamic therapy (PDT) is a promising solution for sitespecific treatment by selectively irradiation tumor regions.7,8 However, the margin of tumor regions is usually not clear; the shape of tumor is irregular; the scattered lights of irradiation are difficult to be controlled. It is not easy to direct light only to microscopic tumors that are surrounded by healthy tissue which is inevitably injured. So, it is very desirable to have “smart” activatable PDT theranostic molecules to differentiate the nidus region from the surrounding healthy tissues, and site-specifically kill tumor cells.9-13 Hypoxia is an important feature of tumor microenvironment due to insufficient oxygen supply in rapid proliferation cancer cells.14-16 It seems a smart strategy to activate PDT efficiency by exploiting hypoxia as the activating factor for PDT. However, tumor hypoxia is also known as the “Achilles’ heel” of

traditional PDT.17,18 Because severe tumor hypoxia hampers therapeutic outcomes of oxygen-dependent PDT, and PDT potentiates hypoxia. Recently, Hanaoka et al reported a mild hypoxia-activatable photosensitizer for PDT based on a selenorosamine dye.19 Under 5% oxygen concentration, the reported “smart” photosensitizer can be activated by azoreductase and give a high PDT efficiency. This work opens a new window to resolve the notorious dilemma between efficient PDT and hypoxia microenvironment. Cancer cells under mild hypoxia are considered as an important target because stemness property of cancer cells is directly related to the mild hypoxia microenvironment.20 Meanwhile, the margin of solid tumor is reasonably the ideal target for PDT, because the gradiently decreased oxygen concentration from outside to inside of the solid tumor makes the outer edge of tumor become the mild hypoxia area. And good PDT efficacy can diminish the tumor volume to make new margin in turn. By this strategy, the severe hypoxic area inside the tumor will be continuously becoming mild hypoxic, which will be very favourable for improving the efficiency of PDT. The “Achilles’ heel” of traditional PDT could be healed. So, it is desirable to develop activatable PDT photosensitizers targeting the margin of solid tumor. However, to the best of our knowledge, such smart PDT photosensitizers working under mild hypoxia microenvironment are very few. Photosensitizers with thermally activated delayed fluorescence (TADF) are emerging triplet-excited-state chromophores without introduction of heavy atoms or transition-metals.

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TADF compounds have attracted many attentions recently for their breakthrough potential in the OLED field.21-23 However, these TADF compounds are usually hydrophobic and not able to be directly used in biological and medical field.24 In our previous work, we developed a new class of TADF photosensitizers based on fluorescein derivatives. These TADF dyes show low-toxicity and were used as chemosensors for cysteine or hypochlorite in living cells.25,26 There are important advantages by using our TADF fluorescein derivatives as theranostic molecular probes. Normally, PDT photosensitizers have a strong ISC process to form triplet excited state. Meanwhile, they cannot keep emitting strong fluorescence or phosphorescence at room temperature. However, our fluorescein derivatives have a narrow energy gap between the singlet excited state (S1) and triplet excited state (T1). So, a delayed fluorescence can be easily observed because of a quick intersystem crossing (ISC) process and a quick reverse ISC process between the two excited states. In other word, these TADF dyes contain a steady S1 and a good fluorescence property. The energy from triplet excited state can be used for PDT therapy by sensitization of oxygen, and the fluorescence from the singlet excited state can be used for optical sensing signal. On the other hand, our TADF fluorescein derivatives have been found to be lysosome targeted, so the singlet oxygen produced in situ on lysosomes could induce lysosomes disruption to achieve better PDT effect. These characters provide them a potential to become better theranostic molecular probes than other PDT photosensitizers. Nitroreductase (NTR) is known as the most studied for overexpression reductases inside tumor tissue induced by hypoxia. In this work, we try to develop smart PDT photosensitizers activated by NTR with our low-toxic TADF fluorescein derivatives. And the smart activation strategy is based on PET mechanism which has often been employed in designing fluorescence probes for NTR. 27-31 The PET process in this work is expected to cage not only the fluorescence decay of singlet excited state, but also the PDT efficiency in which PET competes with the ISC process and weaken the 1O2 production (Scheme 1a). Importantly, mild hypoxia up to 10% oxygen concentration has been found to induce enough NTR to switch on the fluorescence of many probes.29,32-34 Herein, the PDT efficiency is expected to be activated accompanying with the disappearance of the PET process when an enzymatic cleavage reaction occurs which is triggered by NTR induced under mild hypoxia.

Scheme 1. (a) The design strategy for the smart theranostic molecule. (b) The molecular structure of compounds 1-3.

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Experimental Procedures General information. Absorption spectra were measured from a UV-Vis spectrophotometer (Agilent Tech). Fluorescence spectroscopy were recorded on a fluorometer (Agilent Tech). Femtosecond transient absorption spectra were measured by a home-made ultrafast pump–probe setup.35 The pulse duration was 30 fs. Nanosecond fluorescence lifetimes were using the TCSPC technique on HORIBA Jobin Yvon IBN photo counting florescence system with Nano-LED excitation at 456 nm. Microsecond fluorescence lifetimes were using HORIBA Jobin Yvon IBN photo counting florescence system with spectra-LED excitation at 452 nm. Nanosecond timeresolved transient absorption spectra were obtained using an LP920 laser flash photolysis spectrometer (Edinburgh Instruments, UK). Fluorescence imaging was performed by using an OLYMPUS FV-1000 inverted fluorescence microscope with a 60× objective lens. The time-resolved fluorescence images were carried out on a laboratory-use true color time-resolved luminescence microscope. Methods for fluorescence spectroscopy. The stock solutions of compounds were prepared in DMSO and stored at 4 °C in refrigerator. NTR were purchased from Sigma-Aldrich in powder. The stock solution of NTR was prepared in pH 7.4 PBS buffer and preserved in small batches at -20 °C in refrigerator. NADH were purchased from J&K Scientific Company in powder. The NADH was prepared it when it will be used. The compound 1 or 2 (30 L, 10 M) and NADH (30 L, 50 M) were added into pH 7.4 PBS buffer (210 L). To the above mixture, NTR (30 L) was added. The resulting mixture was shaken for 1 second and incubated at 37 °C for certain time. Before fluorescence analysis, CH3CN (2.7 mL) was added to stop the enzyme catalysis. HPLC experiment. HPLC was carried out with H2O (0.3% HAc and 0.3% TEA) and MeOH as eluents by C18 reverse phase chromatography. Gradient elution conditions: H2O/MeOH = 30/70 in the first 20 min, then 0/100 in the next 30 min. Docking calculations. The binding affinity calculations between the compounds and NTR were carried out on AutoDock software (4.2.6 version). The PDB code of NTR was 4DN2. The docking results and figures were obtained from AutoDock and LigPlot +. Singlet oxygen trap experiments. 1, 3Diphenylisobenzofuran (DPBF) was used directly after purchase, in a typical procedure for the detection of singlet oxygen generation, a photosensitizer (10 μM) and DPBF (O.D ~1.0) were mixed in CH3CN. Photo-irradiation were performed by using 590-nm LED (16 mW/cm2). And the distance from the samples and the light was 5 cm. During the entire measurement, the sample needs to be stirred all the time to ensure that the samples were homogenized before absorbance measurements. The absorbance decrease of DPBF at 410 nm was monitored after photoirradiation. Cell and Culture Conditions. HeLa cell line was purchased from the Chenyu biological company (Dalian, China). The culture medium of HeLa cells was Dulbecco's modified Eagle medium (DMEM; Kaiji, Nanjing, China) containing 10% fetal bovine serum (FBS; Sijiqing, Zhejiang, China). The culture conditions of HeLa cells were 37 °C in an atmosphere of 5% CO2 in incubator. HeLa cells were seeded in a 20-mm glass bottom dish from NEST company and incubated for 24 h.


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Anaero Pack-Anaero and Anaero Pack-Micro Aero (Mitsubishi Gas Chemical Company, Japan) were used to create hypoxia condition (1% and 10% oxygen levels). Confocal fluorescence imaging of cells. The HeLa cells were seeded in a glass bottom dish and incubated for 24 h under normoxia condition (oxygen concentrations 21%). Then the cells were incubated for another 6 h at 37 °C under normoxia or hypoxia (oxygen concentrations 10%, and 1%). Under the normoxia or hypoxia atomosphere, the compound 2 (10 M) was added and incubated for another 90 min. The inhibition experiment was done by pre-incubating the cells with dicoumarin (0.2 mM) for 30 min, then incubating with compound 2 for another 90 min under hypoxia (1% O2). The cells were washed with PBS buffer (pH = 7.4) for three times before imaging. Fluorescence images was collected at 610-660 nm upon excitation at 488 nm. Intracellular Singlet Oxygen Detection. DCFH-DA (2, 7dichlorofluorescein diacetate) was used to prove the generation of 1O2 in HeLa cells. The HeLa cells were pre-incubated under normoxia or hypoxia condition (oxygen concentrations 10% and 1%) for 6 h, then incubated with compound 2 (10 M) for 90 min at the same condition, the medium was replaced with fresh medium and balanced in normoxia or hypoxia condition for another 60 min. After that, the cells were subjected to photoirradiation with 590-nm LED (16mW/cm2) under the same condition as before for 10 min. After photoirradiation, the cells were staining with DCFH-DA (10 M) for 20 min. The green channel was excited at 488 nm and collected at 500-520 nm. Subcellular colocalization assay of cells. The HeLa cells were seeded in a glass bottom dish and incubated for 24 h under normoxia condition (oxygen concentrations 21%). The cells were incubated for another 6 h at 37 °C under hypoxia (oxygen concentrations 1%). The subcellular colocalization assay was done under hypoxia atmosphere. In the beginning, the compound 2 (10 M) was added, and the cells were incubated for 90 min. After the culture medium was removed, Lysosome Green (1 M) was added, and the cells were incubated for another 30 min. The green channel was excited at 488 nm and collected at 500-525 nm. The red channel was excited at 488 nm and collected at 610-660 nm. Lysosomes Disruption Assay. The HeLa cells were preincubated under normoxia or hypoxia condition (oxygen concentrations 10%) for 6 h, then incubated with compound 2 (10 μM) for 90 min at the same condition, the medium was replaced with fresh medium and balanced in normoxia or hypoxia condition for another 60 min. After that, the cells were subjected to photoirradiation with 590-nm LED (16mW/cm2) under the same condition as before for 20 min. After photoirradiation, the cells were staining with acridine orange (AO, 5 μM) for 20min. The green channel was excited at 488 nm and collected at 515-545 nm. The red channel was excited at 488 nm and collected at 610-640 nm. Confocal microscopy imaging of cells with AM stainning. The HeLa cells were pre-incubated under normoxia or hypoxia condition (oxygen concentrations 10%) for 6 h, then incubated with compound 2 (10 M) for 90 min at the same condition, the medium was replaced with fresh medium and balanced in normoxia or hypoxia condition for another 60 min. After that, the cells were subjected to photoirradiation with 590-nm LED (16mW/cm2) under the same condition as before for 20 min. After photoirradiation, the cells were assessed using a LIVE/DEAD cell viability assay by staining with Calcein AM

(2 M) for 30 min. The channel of AM was excited at 488 nm and collected at 500-550nm. Cell viability with photoirradiation. The cells were seeded at a density of 1× 105 cells/mL in a 96-well plate for 24 h at 5% CO2 atmosphere, then the cells were incubated for another 6 h under normoxia (oxygen concentrations 21%) or hypoxia (oxygen concentrations 10% or 1%) at 37 °C. After those process, the cells were incubated with different concentration of compound 2, compound 3 or PpIX for 90 min under normoxia or hypoxia condition. Then the medium was removed, and the fresh medium was added, and the plate was balanced in normoxia or hypoxia condition for another 60 min. After that, the cells were irradiated by 590-nm LED lamp (16 mW/cm2) under the same condition as before for 20 min. The cell viability was measured through MTT assays. FACS analysis of annexin V-FITC and PI labeled HeLa cells. The HeLa cells were incubated under normoxia or hypoxia condition (oxygen concentrations 10%) for 6 h and then incubated with compound 2 (10 μM) for 90 min at the same condition, the medium was replaced with fresh medium and balanced in normoxia or hypoxia condition for another 60 min. The cells were subjected to photoirradiation with 590-nm LED (16 mW/cm2) under the same condition as before for 20 min. After photoirradiation, the cells were incubated with FITC (5 µL, KeyGEN BioTECH, Nanjing, China) and PI (5 µL, KeyGEN BioTECH, Nanjing, China) for 30 min. FACS analysis was done to quantify the extent of apoptosis on an Attune NxT Flow Cytometer (Thermo Fisher scientific). Luminescence imaging of mice with endogenous NTR. The luminescence imaging of HeLa tumor mice (n = 4, weight ~25 g) was carried out on a living small animal luminescent imaging analysis system NightOWL II LB983 from Berthold company of Germany. The SCID/BALB/c-nude mice were purchased from Dalian Medical University (Dalian, China, Ethics certificate number is 2018-043). The HeLa cell lines (2×107 cells/mL, 0.1mL) were implanted by subcutaneous injection into the oxter of nude mice to establish HeLa tumor model. The tumor-bearing mice were feed for twenty days when a 1.0-cm diameter tumor model was formed. One group of anaesthetic mice were injected intratumorally with compound 2 (0.1 mM, 100 µL), the other mouse was injected intratumorally with dicoumarin (0.2 mM, 50 µL) for 30 min before the intratumoral injection of compound 2 (0.1 mM, 100 µL). The time-dependent fluorescence changes were collected at every 5 min within 80 min after injection. Then all mice were sacrificed, and the tumors were taken out and embedded with embedding medium. The slices with the thicknesses of 50 μm were obtained by cryosection under -20°C with a microtome CM1860 UV from Leica Company of Germany. The Photocytotoxic effect of tumor-bearing mice. The HeLa tumor-bearing mice were chosen for this research, the HeLa cell lines (2×107 cells/mL, 0.1mL) were implanted by subcutaneous injection into the oxter of nude mice to establish HeLa tumor model. The phototoxicity tests were began when the tumor volume close to 50 mm3, 16 mice were randomly divided into four groups, the first group of mice were intratumoral injected with saline (100 µL) without irradiation (G1), the second group of mice were only irradiated with 590 nm LED (16 mW/cm2) for 30 min on the tumor region (G2), the third group were intratumoral injected with compound 2 (0.1 mM, 100 µL) without irradiation (G3), the last group was intratumoral injected with compound 2 (0.1 mM, 100 µL) at first, and the mice were irradiated with 590-nm LED (16



mW/cm2) for 30 min at tumor region for 40 min after injection (G4). All the mice were treated every three days and last for 4 times, the last 6 days were set as the observation period. The tumor volume and body weights were measured every 3 days during the treatment. The tumor volumes were calculated using the formula V = (a × b2)/2 (a is the longest diameter of tumors, b is the shortest diameter of tumors, and a, b was measured by caliper). When the tumors sizes of the G1 exceeded 800 mm3, the measurements of all groups were stopped, and the mice were sacrificed. H&E staining. The tumors of the four groups of mice were fixed with 10% formaldehyde solution, and then embedded with paraffin to slice the 5 µm sections. The sections were conducted for hematoxylin and eosin (H&E) under standard methods and sent to a microscope.

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after 5 min, which means the activation can be finished very fast (Figure S8). For the sake of accuracy, the 15-minute sensing time was chosen to achieve the working curve for determination of a concentration range of 0 – 0.75 g/mL of NTR (Figure S9). In the chosen sensing conditions, the working curve shows a good linear (R=0.996), and the detection limit was deduced to be 8 ng/mL.

RESULT AND DISCUSSION Design and synthesis of compound 1 and compound 2. Alkylation of the hydroxyl of fluorescein can easily tune the fluorescence property due to the formation of a spiro lactone structure.36-39 Such a smart theranostic molecule 1 was first designed by the above strategy and synthesized from our reported TADF compound 3 (Scheme 1b).40 As expected, compound 1 was synthesized by introduction of the 4nitrophenylchloroformate to the hydroxy of compound 3 (Scheme S1). Meanwhile, a by-product was obtained at the same time, which was further confirmed to be compound 2 by MS and 1H, 13C-NMR, 2D-HMBC. Then compound 2 was resynthesized by a straight way in which compound 3 was treated with 4-nitrobenzyl bromide. Both compound 1 and compound 2 were checked as the smart activatable theranostic molecules in the following NTR response experiments. We also call compounds 1-2 as probes. Spectroscopic responses of compounds 1-2 to NTR. In the beginning, the responses of the probes to NTR were tested in aqueous solutions. The two probes are both in “off” state when compared with strong fluorescence of compound 3 according to their absorption spectra shown in Figure 1a. Their turn-on response towards NTR was investigated by fluorescence spectra (Figure 1b). The sensing mechanisms of these two compounds were proposed as shown in Scheme S2, based on previous reported works.34,41,42 And the sensing mechanisms were verified by HPLC (Figure S1-S2) and MS (Figure S3-S4). During the turn-on process, both compounds 1 and 2 can be transferred into compound 3. Unexpectedly, probe 2 exhibits much faster response and better selectivity than probe 1 (Figure 1c-f). Theoretical calculations were employed to clarify why compound 2 is the better hypoxia-activatable theranostic molecular probe than compound 1. Docking calculations were carried out for the interaction of compound 1 and compound 2 with NTR. Although both 1 and 2 form one hydrogen bonding with NTR (Figure S5), their docking affinities of two compounds have an obvious difference. From Figure S6, the docking energy of compound 1 with NTR is -2.58 ~ -8.62 kcal/mol, while the docking energy of compound 2 is -3.98 ~ -11.36 kcal/mol. The lower binding energy denotes the higher affinity between the compound 2 and NTR.34 After probe 2 was selected as the better photosensitizer for the enzyme activation process, its enzyme catalysis properties were evaluated in detail. The optimum conditions for this enzyme catalysis reaction were found to be 37 °C and pH 7.4 (Figure S7). The time-dependent sensing process began to level off

Figure 1. (a) Normalized absorption and (b) fluorescence emission spectra of compound 1 (10 M), 2 (10 M) and 3 (10 M) in PBS buffer (ex = 488 nm). (c-d) The dynamic changes of fluorescence intensity were recorded for compound 1 and compound 2 response to NTR with different response time. (e-f) The selectivity of fluorescence responses of compound 1 and compound 2 to different kinds of species (1):control, (2): NaCl (50 mM), (3): KCl (50 mM), (4): CaCl2 (50 mM), (5): MgCl2 (50 mM), (6): vitamin C (1 mM), (7): Lys (1 mM), (8): Ser (1 mM), (9): Arg (1 mM), (10): Leu (1 mM), (11): Asp (1 mM), (12): Gly (1 mM), (13): Tyr (1 mM), (14): GSH (10 mM), (15): Hcy (1 mM), (16): Cys (1 mM), (17): KO2 (1 mM), (18): GR (glutathione reductase, 10 g/mL), (19): LEDH (leucine dehydrogenase, 10 g/mL), (20): FDH (formate dehydrogenase, 10 g/mL), (21): DT-diaphorase (5 g/mL), (22): BSA (1 mg/mL), (23): HSA (1 mg/mL), (24): CES1B (100 g/mL), (25): CES1C (100 g/mL), (26): CES2 (100 g/mL), (27): NTR (4 g/mL) in PBS buffer at 37 °C in the presence of probes (10M) and NADH (50 M). The fluorescence intensities were recorded at 635 nm with a 15-min response time after the NTR was added to the mixture (ex = 488 nm). The PDT activation mechanism of compound 2. Since the PET mechanism is expected to cage the fluorescence and the PDT efficiency of the smart theranostic molecule, we move on to clarify how the PET process affects the S1 and T1 state of compound 2. We obtained femtosecond and nanosecond transient absorption spectra for compound 2 and compound 3. As shown in Figure 2a-b and Figure S10 and Table S1, S1 states of compound 2 and compound 3 (600 nm kinetics) decay biexpo-


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nentially. The fast process (32 ps for compound 2, 6 ps for compound 3) is assigned to the vibrational cooling, while the longer decay (690 ps for compound 2, 1270 ps for compound 3), corresponds to S1 state lifetime. This result is consistent with fluorescence lifetime results of compound 2 (0.56 ns) and compound 3 (1.73 ns) (FigureS11 and Table S2). The shorter S1 lifetime of compound 2 compared with compound 3 is due to competition of PET with the fluorescence decay process. These results also explain the stronger fluorescence intensity of compound 3 compared with compound 2, which make compound 2 a good turn-on fluorescence probe for NTR to meet the objective of diagnosis. To make compound 2 become a good PDT photosensitizer to fulfil the goal of treatment, its T1 lifetime should also be triggered by PET process. As shown in Figure 2c-d and Figure S12 and Table S3 (nanosecond transient absorption data), both compound 2 and compound 3 have a longlived flat transient absorption appeared in the 450-650 nm region, but the triplet state lifetime of the compound 2 (1.74 s) is much shorter than compound 3 (16.3 s). It means that PET process in compound 2 does complete with not only the decay of S1, but also the decay of T1. Therefore, the longer T1 of compound 3 than that of compound 2 will result in efficient activation of the PDT efficiency.

ments, the HeLa cells were incubated under three different oxygen concentrations (Figure 3a-c). With the decrease of the O2 concentration from 21% to 1%, the fluorescence intensity of the tumor cells was increased correspondingly. And the intracellular inhibition experiments with dicoumarin, a known inhibitor, 34,46,47 support that the enhanced fluorescence was due to the endogenous NTR (Figure 3d). The two-photon imaging data further support our above explanation (Fig S14-15) and offer compound 2 a two-photon PDT potential for deeper tissue treatment.7,8,48,49 These results prove the endogenous NTR can catalyze the enzymatic cleavage reaction of compound 2 to compound 3 in living tumor cells. Furthermore, the longer T1 activation process can be visually captured by time-resolved luminescence imaging. According to our previous work, the longliving triplet excited state in our fluorescein derivatives can bring out the long TADF lifetime by RISC process.25,40 The lifetime of compound 3 is 22.14 s in deaerated ethanol, much longer than the luminescence lifetime of compound 2. (Figure S16 and Table S4). As shown in Figure 3e, the activated longliving luminescence was captured successfully by time-resolved luminescence imaging experiments (delayed time 33 s) accompanying the enzymatic cleavage reaction of compound 2 to compound 3 under hypoxia conditions.

After that, the PDT abilities of compound 2 and 3 were checked whether singlet oxygen can be efficiently generated by the two compounds under photoirradiation. We used 1, 3-diphenylisobenzofuran (DPBF) as a singlet oxygen trap to check the ability of production singlet oxygen of compound 2 and compound 3 in acetonitrile.43-45 As shown in Figure S13, the rate of singlet oxygen generation by compound 3 was much faster than by compound 2. This result indicates that the PDT performance of compound 2 could be efficiently activated according to the different ability of 1O2 production between compounds 2 and 3.

Figure 2. (a-b) Femtosecond transient absorption spectra of compound 2 (500 M) and 3 (100 M) in deaerated ethanol at different decay times (ex = 450 nm). (c-d) Nanosecond transient absorption spectra of compound 2 (10 M) and 3 (10 M) in deaerated ethanol at different decay times (ex = 532 nm). Fluorescence and PDT activation of compound 2 in living cells. Since the response of compound 2 to NTR has been well investigated in aqueous solutions mainly containing PBS buffer, we try to assess the response of compound 2 to NTR in living HeLa cells by one-photon and two-photon confocal fluorescence imaging. In the one-photon fluorescence imaging experi-

Figure 3. (a-c) Confocal fluorescence images of HeLa cells incubated with compound 2 (10 M) for 90 min under different oxygen concentration conditions (a: 21%; b: 10%; c: 1%). (d) Confocal fluorescence images of inhibition experiments with dicoumarin (HeLa cells were pre-incubated with dicoumarin (0.2 mM) for 30 min, then incubated with compound 2 (10 M) for 90 min under 1% oxygen concentration). (e) Time-resolved fluorescence images of HeLa cells incubated with compound 2 (10 M) for 90 min under 1% oxygen concentration. The scale is 30 m.



Then the NTR-activated PDT efficiency was checked in living tumor HeLa cells by MTT assay under three different oxygen concentrations. As shown in Figure 4a, the PDT efficiency of the traditional photosensitizer PpIX is highly dependent on oxygen concentration. Server hypoxia at 1% oxygen concentration will dramatically hamper the PDT efficiency. Interestingly, the efficiency of PDT at 10% oxygen concentration remains similar to that at ambient oxygen concentration, which is consistent with others’ work.50 The similar results were achieved for compound 3 (Figure S17). It means the PDT performance of PpIX and compound 3 keep almost the same level of photocytotoxicity under between 10% and 21% oxygen concentration. However, the PDT efficiency of compound 2 at 10% oxygen concentration are much higher than at 21% (Figure 4b), and the half-maximal inhibitory concentration of compound 2 (IC50 = 5.91 M) was even lower than that of PpIX (IC50 = 6.46 M) at 10% oxygen concentration, which means our activation strategy is successful. The success of PDT activation process should attribute that mild hypoxia at 10% oxygen concentration can induce enough NTR to trigger the transfer from compound 2 to 3. It is noteworthy in the confocal fluorescence imaging experiment that mild hypoxia condition (10% oxygen concentration) was found to be able to induce enough NTR to switch on the fluorescence.(Figure 3b) It means the endogenous NTR can catalyze the enzymatic cleavage reaction of compound 2 to compound 3 in living tumor cells even under 10% oxygen concentration, which leads to efficient PDT under mild hypoxia condition.

Figure 4. (a-b) The cell viabilities of HeLa cells based on MTT assay of PpIX and compound 2 under 590 nm LED light irradiation. (c) The AM staining of compound 2 treated HeLa cells without (c1, c3) or with (c2, c4) irradiation in normoxia (c1-c2) or hypoxia (10%, c3-c4) condition. (d) The FACS of annexin V-FITC and PI labeled HeLa cells without (d1, d3) or with (d2, d4) irradiation in normoxia (d1-d2) or hypoxia (10%, d3-d4) condition. The scale is 30 m.

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oxygen concentration gives the best 1O2 generation yield according to the bright green fluorescence intensity. In addition, the activated the PDT efficiency by mild hypoxia of 10% oxygen concentration was confirmed by using confocal fluorescence image and the fluorescence-activated cell sorting (FACS) statistical analysis. By comparing images c1 with c3 for no-irradiation cells in Figure 4c, we found that the mild hypoxic cells have a similar good survival state with the normoxic cells without photoirradiation, considering that Calcein AM is a living-cells staining dye with green fluorescence.26,51-54 By comparing images c1 with c2 for normoxic cells, we found that the photoirradiation did not hurt the cells. Since no green fluorescence was observed in image c4 for the mild hypoxic cells with photoirradiation, these results indicate the PDT efficiency can be activated only when the two demands of photoirradiation and mild hypoxia are met. According to the FACS statistical analysis shown in Figure 4d, the percentage of apoptosis (46.6%) under mild hypoxia condition, was much higher than that (0.05%) under normoxia condition for the HeLa cells with photoirradiation. The FACS statistical analysis not only confirmed that the PDT efficiency of compound 2 can be activated by mild hypoxia, but also suggested that the PDT induced cells death is via apoptosis, not necrosis.55,56 To further investigate the therapeutic mechanism of compound 2-mediated PDT, a subcellular colocalization experiment of compound 2 was performed by co-staining with the organelle-specific fluorescent dye Lysosome Green, a commercial lysosome tracker.51,57,58 As showing in Figure 5, the Pearson's correlation coefficient is up to 0.93, which means the compound with red fluorescence is localized in the lysosome. According to our previous work,40 compound 3 was found to localize in the lysosome. These results prove that the endogenous NTR can catalyze the enzymatic cleavage reaction of compound 2 to compound 3 in living tumor cells, and the formed compound 3 can localize in the lysosome. Based on these results, we speculate that the cell apoptosis was because the intracellular lysosomes were destroyed by the 1O2 generated in situ. To prove the intracellular lysosomes lysis, acridine orange (AO) was used as the indicator for lysosomal integrity.57,59 As shown in Figure 6, the red fluorescence which represents the integrity of lysosomes disappeared only in the group of the cells irradiated by 630-nm LED under 10% oxygen concentration. This result indicates that the lysosomes were destroyed by the 1O2 generated in situ.

Figure 5. Subcellular colocalization assay of HeLa cells. The cells were incubated with compound 2 (10 M) for 90 min, then incubated with Lysosome Green (1 M) for 30 min. (a1) the image of Lysosome Green; (a2) the image of compound 2; (a3) the merged images of a1, a2 and bright-field; (a4) the correlation plot of Lysosome Green and compound 2.

To make sure the cells death was caused by the generation of O2, we use 2, 7-dichlorodihydrofluorescein diacetate (DCFHHA) as a 1O2 probe to further prove the intracellular 1O2 generation by confocal imaging.51 As shown in Figure S18, the green fluorescence suggests the 1O2 was generated under hypoxia condition with irradiation. At the same time, it is obvious that 10% 1


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Figure 6. The acridine orange (AO) staining confocal fluorescence images of HeLa cells incubated with compound 2 (10 M) for 90 min. (a1) HeLa cells were treated under normoxia condition without irradiation; (a2) HeLa cells were treated under normoxia condition with irradiation; (a3) HeLa cells were treated under hypoxia condition without irradiation; (a4) HeLa cells were treated under hypoxia condition with irradiation; (b1b4) the bright field images correspond to a1-a4. The scale is 30 m. Fluorescence and PDT activation of compound 2 in tumor bearing mice. After the NTR-activated PDT performance of compound 2 was verified in the living cells, we try to explore how the activation can be fulfilled in vivo. Anesthetized SCID/BALB/c-nude mice were chosen as mammal model. The fluorescence response of the compound 2 toward endogenous NTR was tested in HeLa tumor-bearing mice. After the tumor model formed with a 1.0-cm diameter, one group was injected with the compound 2, and the other group was injected with compound 2 and dicoumarin. The time-dependent fluorescence images and intensity were collected every 5 min after injection. As shown in Figure 7, there was an obvious distinction in the fluorescence brightness between these two groups due to the inhibition effect of dicoumarin. These results indicate that endogenous NTR in the tumor area can efficiently switch on the fluorescence of the compound 2. And the fluorescence intensity reached maximum around 30 min after injection and levelled off until 80 min, which is still a quick response time considering the hysteresis effect due to the diffusion process of the sensing probe. From the confocal images of the tumor slices dissected at 80 mins (Figure S19), the distinct red fluorescence is observed in the whole image. This result indicates that the compound 2 was distributed evenly inside the whole tumor after 80 min, which gives PDT a good working time zone. Meanwhile, since the extent of hypoxia should become mild at the edge of the solid tumor, this result also confirms NTR can be induced even under mild hypoxia to trigger the transfer from compound 2 to 3. And it means that in vivo PDT will have a good oxygen concentration around 10% to achieve a high efficiency.

Figure 7. Fluorescence imaging of endogenous NTR activities in HeLa tumor based SCID/BALB/c-nude mice by intratumor injection with compound 2 (0.1 mM, 100 µL). (a) Time-dependent fluorescence imaging of mouse tumor model. (b) Time-dependent fluorescence quantification of the tumor sections in (a). Error bars indicate SD (n = 2). At last, we checked the high PDT performance of compound 2 in HeLa tumor-bearing mice. The mice were randomly divided into four groups. As shown in Figure 8a-b, the tumors growth of mice under the combination conditions of existing compound 2 and irradiation were completely inhibited, while all the control mice of the other three groups exhibited no obvious therapeutic effect. Besides, no significant changes could be found from the weights of all these four groups of mice (Figure 8c), and there was no obvious change in the pericancerous skin tissue before and after treatment as shown in Figure S20. It means that the side effect of both compound 2 and irradiation were negligible. To further verify the PDT efficiency in mice, the tumor sections were stained with hematoxylin and eosin (H&E) after the mice were sacrificed. As shown in Figure 8d, significant apoptosis can only be observed in the positive tumor sections. All these results support that compound 2 has high activatable phototoxicity and low side effect, which make it possible to differentiate the nidus region from the surrounding healthy tissues and site-specifically inactivate tumor cells.



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traditional photosensitizer. The excellent PDT effect was associated with its lysosomal targeting ability. Besides the NTR-activatable theranostic property, compound 2 also possesses many other advantages such as two-photon excitation, large Stokes shift, short response time, high specificity and sensitivity. Importantly, the strategy of targeting the mild hypoxic cells in the edge of tumor tissue could heal the “Achilles’ heel” of traditional PDT. We believe this smart theranostic molecule has high potential to be applied in clinical investigation as a theranostic anti-cancer prodrug.

ASSOCIATED CONTENT Supporting Information Synthesis of compounds 1-3, supporting data for activation mechanism, docking calculations of compounds 1-2 with NTR, fluorescence spectra, luminescence lifetime, confocal fluorescence imaging. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Fengling Song. E-mail: [email protected], [email protected].

ORCID Fengling Song: 0000-0001-5305-676X

ACKNOWLEDGMENT Figure 8. The PDT efficiency in HeLa tumor mice with 590 nm LED (16 mW/cm2). (a) The photos of the four groups’ tumorbearing mice. (b) The changes of tumor volumes of the four groups. Error bars indicate SD (n = 4), date was determined by using Sidak’s multiple comparison test. (c) The changes of the weight of the mice through the Phototoxicity test. (d) H&E staining of tumor tissue sections (from left to right, 1: saline, 2: Irradiation, 3: compound 2 (0.1 mM, 100 µL) without irradiation, 4: compound 2 (0.1 mM, 100 µL) with irradiation). The scale is 50 m.

This work was supported financially by the National Natural Science Foundation of China (21877011，21576038, 21421005), the Fundamental Research Funds for the Central Universities of China (DUT16TD21), DUT startup grant (G.G.G.), DUT basic research funding (DUT18GJ205) and Science Program of Dalian City (2015J12JH207). This work was also supported by Supercomputing Center of Dalian University of Technology.

REFERENCES

Conclusion In summary, we have synthesized two smart activatable theranostic molecules 1 and 2 based on compound 3, a TADF fluorescein derivative. Compound 2 has better selectivity and response rate than compound 1 when sensing NTR. Importantly, the PDT efficiency of the selected compound 2 can be efficiently activated by mild hypoxia microenvironment of 10% oxygen concentration. During the activation, compound 3 can be generated in situ from compound 2. The formed compound 3 has a long triplet state lifetime (16.3 s), which leads to efficient production of 1O2. The NTR-activation process was verified to be done under mild hypoxia in living HeLa cells and in vivo animals. To our delight, it was found that the PDT efficiency of compound 2 under hypoxia condition (10% oxygen concentration) was much better than under normoxia (21% oxygen concentration). In fact, compound 2 under mild hypoxia has a high PDT efficiency, even betten than PpIX, a

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11 ACS Paragon Plus Environment

Nitroreductase-activatable theranostic molecules with high PDT

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