Enzyme-Responsive Double-Locked Photodynamic Molecular Beacon for Targeted Photodynamic Anticancer Therapy

An advanced photodynamic molecular beacon (PMB) was designed and synthesized, in which a distyryl boron dipyrromethene (DSBDP)-based photosensitizer and a Black Hole Quencher 3 moiety were connected via two peptide segments containing the sequences PLGVR and GFLG, respectively, of a cyclic peptide. These two short peptide sequences are well-known substrates of matrix metalloproteinase-2 (MMP-2) and cathepsin B, respectively, both of which are overexpressed in a wide range of cancer cells either extracellularly (for MMP-2) or intracellularly (for cathepsin B). Owing to the efficient Förster resonance energy transfer between the two components, this PMB was fully quenched in the native form. Only upon interaction with both MMP-2 and cathepsin B, either in a buffer solution or in cancer cells, both of the segments were cleaved specifically, and the two components could be completely separated, thereby restoring the photodynamic activities of the DSBDP moiety. This PMB could also be activated in tumors, and it effectively suppressed the tumor growth in A549 tumor-bearing nude mice upon laser irradiation without causing notable side effects. In particular, it did not cause skin photosensitivity, which is a very common side effect of photodynamic therapy (PDT) using conventional “always-on” photosensitizers. The overall results showed that this “double-locked” PMB functioned as a biological AND logic gate that could only be unlocked by the coexistence of two tumor-associated enzymes, which could greatly enhance the tumor specificity in PDT.


Figure S13
Bright field, fluorescence, and the merged images of HeLa and HEK-293 cells after incubation in a serum-free medium in the absence or presence of SB-3CT (10 μM) and/or CA-074 Me (25 μM) for 2 h, and then with 1 (2 μM) for 1 h, followed by post-incubation in the medium for a further 6 h or incubation with 14 (2 μM) for 1 h, followed by post-incubation in the medium for a further 6 h.

Figure S18
ESI mass spectrum of 3.

Figure S19
ESI mass spectrum of 5.

Figure S20
ESI mass spectrum of 8.

Figure S21
ESI mass spectrum of 11.

Figure S22
ESI mass spectrum of 12.

Figure S23
ESI mass spectrum of 13.

Figure S24
ESI mass spectrum of 1.

Figure S25
ESI mass spectrum of 14.

Experimental Section
General All the reactions were performed under an atmosphere of nitrogen. CH2Cl2, DMF, and tetrahydrofuran (THF) were purified using an INERT solvent purification system. All other solvents and reagents were of HPLC or reagent grade and used as received. and steady-state fluorescence spectra were taken on a Shimazu UV-1800 UV-Vis spectrophotometer and a HORIBA FluoroMax-4 spectrofluorometer, respectively.

Preparation of 3
Peptide 3 was prepared manually using a modified Fmoc SPPS protocol as shown in Scheme S1. The Fmoc protecting group on the rink amide resin was first removed using 20% piperidine in DMF. An excess of Fmoc-protected amino acids (4 equiv) or Fmoc-Lys(N3)-OH, HATU (4 equiv), and DIPEA (8 equiv) in DMF were used for each coupling at room temperature. After the final coupling and Fmoc deprotection, the resin was treated with a mixture of 95% TFA, 2.5% TIPS, and 2.5% water for 1 h to remove the protecting groups and release the peptide.
The resin was removed by filtration, and the filtrate was precipitated upon addition of diethyl ether. After centrifugation, the supernatant was removed. The solid was redissolved in DMSO and then precipitated by the addition of diethyl ether. The crude product was lyophilized and then purified by reverse-phase HPLC. The conditions used were set as follows: solvent A = 0.1% TFA in acetonitrile and solvent B = 0.1% TFA in deionized water. Elution gradient: 0% A + 100% B in the first 5 min, changed to 100% A + 0% B in 30 min, maintained at 100% A for 5 min, changed to 0% A + 100% B in 10 min, and maintained at 100% B for further 10 min.
A flow rate of 1 or 3 mL min -1 was used for analytical or preparative purpose, respectively.

Preparation of 5
Peptide 5 was prepared according to the procedure described for 3 (Scheme S2). After lyophilization of the precipitated peptide, the crude product was purified by reverse-phase HPLC with the aforementioned conditions. HRMS (ESI): m/z calcd for C66H123N29O20S2 [M+4H] 4+ 426.4729, found 426.4723.
The conditions used were set as follows: solvent A = 0.1% TFA in acetonitrile and solvent B = 0.1% TFA in deionized water.

Preparation of 1
BCN-substituted BHQ-3 11 (1.3 mg, 1.9 μmol) was added into the above HPLC-purified 12 in a mixture of acetonitrile and water. After complete consumption of 12 as indicated by reverse-S12 phase HPLC, the target product 1 was isolated with the same conditions described for the purification of 12 and then lyophilized to give a dark bluish-green solid (

Preparation of 14
According to the above procedure using BCN-substituted BHQ-3 11 (1.2 mg, 1.8 μmol) and the HPLC-purified 13 as the starting materials, conjugate 14 was purified and then lyophilized to give a dark bluish-green solid (1.1 mg, 10% for the two-step reaction

Monitoring the Course of the Formation of 1 and 14
The course of the formation of 1 and 14 was monitored by LC-MS. Reverse-phase LC-MS analysis was performed on a XBridge BEH300 C18 column (5 μm, 4.6 mm × 150 mm) using a Waters ACQUITY Arc system equipped with a Waters 2998 photodiode array detector, a Waters 2475 fluorescence detector, and a Waters SQD 2 mass spectrometer. The conditions used were set as follows: solvent A = 0.01% formic acid in acetonitrile and solvent B = 0.01% formic acid in deionized water. Elution gradient: 20% A + 80% B in the first 5 min, changed to 100% A + 0% B in 30 min, maintained at 100% A for 5 min, changed to 20% A + 80% B in S13 10 min, and maintained at 20% A + 80% B for further 10 min. A flow rate of 0.8 mL min -1 was used for analytical purpose.

Determination of Fluorescence Quantum Yields
The fluorescence quantum yields (ΦF) of the samples were determined by the equation: where W and Iabs are the DPBF photobleaching rate and the rate of light absorption, respectively. R6

MMP-2 and Cathepsin B-Responsive Spectroscopic Studies
Compound 1 or 14 was dissolved in DMF to form a 1 mM stock solution, which was then All of the solutions were stirred continuously at 37 °C. Their electronic absorption and fluorescence spectra were monitored over a period of 25 h.

MMP-2-and Cathepsin B-Responsive Singlet Oxygen Generation Studies
According to the above procedure, 1 and 14 were treated with MMP-2 and cathepsin B. The solutions were then mixed with a solution of DPBF in DMF (3 mM, 3 μL), giving a final concentration of 30 M. The mixtures were irradiated with red light coming from a 100 W S15 halogen lamp after passing through a water tank for cooling and a color glass filter (Newport) cut-on at λ = 610 nm. The decay of DPBF at λ = 417 nm was monitored with a spectrophotometer during the irradiation period.

Monitoring the Reactions of 1 with MMP-2 and/or Cathepsin B
According to the above procedure, 1 and 14 were treated with MMP-2 and cathepsin B. After a period of 25 h, MeOH was added into the solutions for precipitating the enzymes. After filtration, the filtrate was analyzed by LC-MS using the aforementioned conditions. supplemented with FBS (10%) and penicillin-streptomycin (100 units mL −1 and 100 μg mL −1 , respectively). All the cells were grown at 37 °C in a humidified 5% CO2 atmosphere.

Confocal Fluorescence Microscopic Studies
Approximately 2 × 10 5 cells in the corresponding medium (2 mL) were seeded on a confocal dish and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. Compound 1 or 14 (20 nmol) was first dissolved in DMF (20 μL) to prepare a stock solution of 1 mM, which was diluted with a serum-free medium to 1 μM. For the enzyme-responsive studies, the cells were incubated in the medium (1 mL) with or without SB-3CT (10 μM) and/or CA-074 Me (25 μM) for 2 h. The cells were then incubated with 1 (2 μM) in the same medium for a further 1 h. As a negative control, the cells were simply incubated with 14 (2 μM) in the medium (1 mL) for 1 h. After being rinsed with PBS twice, the cells were further incubated in the medium for 6 h.
The cells were then rinsed with PBS twice before being examined with a Leica TCS SP8 highspeed confocal microscope equipped with a 638 nm laser. The fluorescence was monitored at 650−750 nm. The images were digitized and analyzed using a Leica Application Suite X software.

Flow Cytometric Studies
Approximately 2 × 10 5 cells in the corresponding medium (2 mL) were seeded on a 6-well plate and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. After the S17 treatments as described above, the cells were rinsed with PBS twice and then harvested by 0.25% trypsin-EDTA (Invitrogen, 0.3 mL) for 5 min. The activity of trypsin was quenched with a serum-containing medium (0.7 mL), and the mixture was centrifuged at 1500 rpm for 3 min at room temperature. The pellet was washed with PBS (1 mL) and then subject to centrifugation. The cells were suspended in PBS (1 mL) and the intracellular fluorescence intensities were measured using a BD FACSVerse flow cytometer (Becton Dickinson) with 1 × 10 4 cells counted in each sample. The compounds were excited by an argon laser at 640 nm and the emitted fluorescence was monitored at 720−840 nm. The data collected were analyzed using the BD FAC-Suite. All experiments were performed in triplicate.

Subcellular Localization Studies
Approximately 2 × 10 5 A549 cells in DMEM (2 mL) were seeded on a confocal dish and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. A stock solution of PMB 1 (1 mM) was prepared as described above, which was then diluted to 1 μM with a serum-free medium. The medium was removed and the cells were rinsed with PBS twice. The cells were first incubated with 1 (1 μM) for 1 h. After being rinsed with PBS twice, the cells were incubated in a serum-free medium for 6 h. The cells were rinsed with PBS twice, followed by staining with LysoTracker Green DND-26 (Thermo Fisher Scientific Inc., L7526) (2 μM for 30 min), MitoTracker Green FM (Thermo Fisher Scientific Inc., M7514) (0.2 μM for 15 min), S18 or ER-Tracker Green (Thermo Fisher Scientific Inc., E34251) (1 μM for 15 min) in a serumfree medium at 37 °C. The solutions were then removed, and the cells were rinsed with PBS twice before being examined with a Leica TCS SP8 high-speed confocal microscope equipped with a 488 nm laser and a 638 nm laser. All the trackers were excited at 488 nm and their fluorescence was monitored at 500−570 nm, while the DSBDP moiety was excited at 638 nm and its fluorescence was monitored at 650−750nm. The images were digitized and analyzed using a Leica Application Suite X software.

Intracellular ROS Generation Studies
Approximately 2 × 10 5 A549 cells in DMEM or HeLa cells in RPMI 1640 medium (2 mL) were seeded on a confocal dish and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. A stock solution of 1 or 14 (1 mM) was prepared as described above, which was diluted to 1 μM with a serum-free medium. The cells were treated as described above. After being rinsed by PBS twice, the cells were incubated with H2DCFDA in PBS (10 µM, 1 mL) at 37 °C for 30 min. The cells were rinsed and refilled with PBS (1 mL) before being irradiated (λ > 610 nm, 23 mW cm -2 , 14 J cm -2 ) at ambient temperature. The cells were examined with a Leica TCS SP8 high-speed confocal microscope equipped with a solid-state 488 nm laser. DCF, the oxidized product of H2DCFDA, was excited at 488 nm and its fluorescence was monitored S19 at 500−550 nm. The images were digitized and analyzed using a Leica Application Suite X software.

Photocytotoxicity Assay
Approximately 2 × 10 4 cells per well (100 L) in the corresponding medium were inoculated in 96-well plates and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. A stock solution of 1 or 14 (1 mM) was prepared as described above, which was diluted with a serum-free medium to various concentrations. The cells, after being rinsed with PBS twice, were treated with or without SB-3CT (10 μM) and/or CA-074 Me (25 μM) in the medium (100 μL) for 2 h. The cells were further incubated with 100 μL of different concentrations of 1 or 14 for 1 h at 37 °C under 5% CO2. After being rinsed with PBS twice, the cells were incubated in the culture medium (100 μL) for 6 h. The cells were then rinsed with PBS and re-fed with 100 μL of a serum-containing medium before being irradiated (λ > 610 nm, 23 mW cm -2 , 28 J cm -2 ) at ambient temperature. Cell viability was determined by means of a colorimetric MTT assay. R7 After irradiation, the cells were incubated at 37 °C under 5% CO2 overnight. After incubation, the medium was removed and the cells were rinsed with PBS twice. A MTT (Sigma) solution in PBS (3 mg mL -1 , 50 μL) was added to each well followed by incubation for 4 h under the same environment. DMSO (100 μL) was then added to each well. Solutions in all the wells were mixed until homogenous. The absorbance at 490 nm of each well on the plate was S20 taken by a microplate reader (Tecan Spark 10M) at ambient temperature. The average absorbance of the blank wells, which did not contain the cells, was subtracted from the readings of the other wells. The cell viability was then determined by the equation: where Ai is the absorbance of the ith datum (i = 1, 2, …, n), Acontrol is the average absorbance of the control wells in which the compound was absent, and n (= 4) is the number of data points.

In Vivo Imaging
Female Balb/c nude mice (20-25 g) were obtained from the Laboratory Animal Services Centre at The Chinese University of Hong Kong. All animal experiments were approved by the Animal Experimentation Ethics Committee of the City University of Hong Kong. The mice were kept under a pathogen-free condition with free access to food and water. A549 cells (1 × 10 7 cells in 200 μL) were inoculated subcutaneously at the back of the mice. Once the tumors had grown to a size of 80-100 mm 3 , 1 or 14 (20 nmol) dissolved in 20 μL of distilled water containing 7.5% DMSO and 0.5% Tween 80 (v/v) was injected to the tumor-bearing mice through intratutormal injection. In vivo fluorescence imaging was performed before and after the injection at different time points up to 24 h with an Odyssey infrared imaging system (excitation wavelength = 680 nm, emission wavelength ≥ 700 nm). The images were digitized S21 and analyzed using an Odyssey imaging system software (no. 9201-500). Four mice were used for each compound.

In Vivo PDT
A549 tumor-bearing nude mice were prepared as described above. The length and width of the tumor were measured using a micrometer digital caliper (SCITOP Systems). The tumor volume (mm 3 ) was calculated using the formula: tumor volume = (length × width 2 )/2. Once the tumors had grown to a size of 80-100 mm 3 , 1 or 14 (20 nmol) dissolved in 20 μL of distilled water containing 7.5% DMSO and 0.5% Tween 80 (v/v) was injected to the tumor-bearing mice through intratumoral injection. At 6 h post-injection, the tumor was illuminated with a diode laser (Biolitec Ceralas) at 680 nm operated at 0.24 W. Illumination on a circular spot with a diameter of 1.0 cm (fluence rate = 0.3 W cm -2 ) for 10 min led to a total fluence of 180 J cm -2 .
The tumor size of the nude mice was monitored periodically for the next 14 days. The tumor volumes were also compared with a PBS control group of mice without the light treatment.
Four mice were used for each group. On Day 14 after the PDT treatment, the mice were sacrificed, and the tumor and major organs were harvested. The tissues were fixed with 4% paraformaldehyde, dehydrated in an alcohol series, mixed with solutions of xylene and paraffin, and embedded in paraffin. The tissue sections were prepared by a Leica RM2235 microtome.

S22
After the H&E staining, the tissues were examined with a Carl Zeiss PALM inverted microscope.

In Vivo Photodynamic Effect on Skin
Female Balb/c nude mice (20-25 g) were injected with a solution of 1 or 6 (20 nmol) in 200 μL of distilled water containing 3% DMSO and 0.5% Tween 80 (v/v) through the tail vein. In vivo fluorescence imaging was performed before and after the injection at different time points up to 24 h with 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 (no. 9201-500). Three mice were used for each compound. To study the photodynamic effect of these two compounds on the skin of the mice, a region of the skin was illuminated with a diode laser (Biolitec Ceralas) at 680 nm operated at 0.24 W at 6 h postinjection of these compounds. Illumination on a circular spot with a diameter of 1.0 cm (fluence rate = 0.3 W cm -2 ) for 10 min led to a total fluence of 180 J cm -2 . After 24 h, the mice were sacrificed, and then the skin tissues were harvested. They were fixed with 4% paraformaldehyde, dehydrated in an alcohol series, mixed with solutions of xylene and paraffin, and then embedded in paraffin. The tissue sections were prepared by a Leica RM2235 microtome. After the H&E staining, the tissues were examined with a Nikon Eclipse Ni-E upright fluorescence microscope. For the TUNEL staining assay, the skin tissues were dewaxed S23 and rehydrated in an alcohol series and then incubated with a Proteinase K solution (20 μg mL -1 ) at room temperature for 30 min. After being rinsed with PBS three times, the tissue slides were stained with 50 μL of Nucleotide Mix (a mixture of terminal deoxynucleotidyl transferase and cyanine 3-labeled deoxyuridine triphosphate) of the one-step TUNEL assay kit (Beyotime, Shanghai, China), and then the nuclei were stained with Hochest 33342 (10 μM) for 1 h at room temperature. After being rinsed with PBS for three times, the tissue slides were examined with a Zeiss LSM 880 NLO laser scanning microscope equipped with two solid-state 405 and 561 nm lasers. Hoechst 33342 was excited at 405 nm and its fluorescence was monitored at 410−500 nm, while the TUNEL staining cyanine 3 dye was excited at 561 nm and its fluorescence was monitored at 570−620 nm. The images were digitized and analyzed using a Zen software.

Statistical Analysis
Data shown on figures are presented as the means with the SEM or SD. The data were analyzed using the Student's t-test with p values < 0.05 considered as significant; *p < 0.05; **p < 0.01; and ***p < 0.001. Statistical calculations were performed using a Microsoft Excel spreadsheet   Figure S24. ESI mass spectrum of 1. The insets show the experimental and simulated isotopic patterns for the [M+4H] 5+ ion.