Cucurbit[8]uril Regulated Activatable Supramolecular Photosensitizer

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Cucurbit[8]uril Regulated Activatable Supramolecular Photosensitizer for Targeted Cancer Imaging and Photodynamic Therapy Xiao-Qiang Wang, Qi Lei, Jing-Yi Zhu, Wen-Jing Wang, Qian Cheng, Fan Gao, Yun-Xia Sun, and Xian-Zheng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07507 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 12, 2016

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ACS Applied Materials & Interfaces

Cucurbit[8]uril Regulated Activatable Supramolecular Photosensitizer for Targeted Cancer Imaging and Photodynamic Therapy Xiao-Qiang Wang, Qi Lei, Jing-Yi Zhu, Wen-Jing Wang, Qian Cheng, Fan Gao, Yun-Xia Sun,* and Xian-Zheng Zhang* Key Laboratory of Biomedical Polymers of Ministry of Education, the Institute for Advanced Studies & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China KEYWORDS supramolecular chemistry • activatable photosensitizer • singlet oxygen • photodynamic therapy • anticancer

ABSTRACT: Activatable photosensitizers (aPSs) have emerged as promising photodynamic therapy (PDT) agents for simultaneous imaging and selective ablation of cancer. However, traditional synthetic aPSs are limited by complex design and tedious synthesis. Here, A aPS regulated by cucurbit[8]uril (CB[8]) for targeted cancer imaging and PDT is reported. This system is based on the host-guest interaction between biotinylated toluidine blue (TB-B) and CB[8] to form 2TB-B@CB[8]. Moreover, a facile strategy to turn off/on the fluorescence and photodynamic activity of TB-B is developed through the reversible assembly/disassembly of 2TB-B@CB[8]. This established system can achieve selective accumulation in tumour, light up cancer imaging and enhanced anticancer behaviour. Therefore, this work provides a novel and

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promising strategy for the aPS build via simply and facile regulation of supramolecular chemistry.

1. INTRODUCTION Photodynamic therapy (PDT) has emerged as a promising cancer-therapy modality for clinical use.1-3 In PDT, photosensitizers (PSs) are the key component which can transfer the energy of light to the surrounding oxygen. Followed by the generation of toxic reactive oxygen species (ROS), especially singlet oxygen (1O2), which can induce cell apoptosis or necrosis. Compared with traditional chemotherapy, PDT exhibits advantages of minimally invasive nature and high spatiotemporal precision.4 However, currently available PSs cannot adjust themselves for the selective cancer cells imaging and killing, they are not smart enough to meet the demand of personalized treatment. To tackle these drawback, activatable photosensitizers (aPSs) as personalized medicine have been developed in recent years.5-13 aPSs are pre-quenched PSs that can be activated when specific disease-related triggers appear. Generally, the design of aPSs is based on the principle of blocking the energy transfer pathways between PSs and oxygen after light irradiation, and the most used strategy is covalent anchoring quenchers or energy accepters to the vicinity of PSs with cleavable linkers.5-13 As a result, aPSs can help early diagnose of cancer, selectively deliver PSs in tumour, real-time monitor PSs release, further reduce side effect and improve therapeutic efficacy.5-13 However, this covalent decoration strategy to architect aPSs suffers from problems such as complex molecular design, tedious synthesis and uneconomic clinical use. In the view of supramolecular chemistry, non-covalent interactions may offer more facile strategy to build aPSs. The dynamic nature of non-covalent interactions enables supramolecular


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complexes to assemble and disassemble through a range of stimuli-responsive processes.14-21 Cucurbit[n]uril (CB[n]) family are widely studied macrocyclic host molecules.22-24 Due to their molecular recognition property, extraordinary binding affinity, high selectivity, rich complementary guests pool and multiple binding models, CB[n]s can be used as facile tools in the building of functional biomedical materials.25-28 For example, CB[7] as gatekeeper to regulate the therapeutic efficacy of gold nanoparticles has been reported by Rotello et al,29 in their following work, the biorthogonal catalysis ability of transition mental catalysts could also be reversible controlled by this CB[7] gatekeeper.30 Furthermore, CB[n]s also exhibit ability to directly alter the physical or chemical property of some dyes.31 For example, a series of tricyclic basic dyes such as toluidine blue, methylene blue and acridine orange can bind with CB[7] and CB[8] with changed fluorescent emission. More interestingly, the binding with CB[7] enhances the fluorescence whereas the binding with CB[8] leads to the quenching effect.32 In addition to the ability to alter the fluorescent property, we further hypothesize that the host-guest chemistry of CB[n] may also affect the photodynamic activity of the dyes, which is highly desirable for the design of novel aPSs regulated by supramolecular chemistry. The phenothiazinium photosensitizers, including toluidine blue (TB), methylene blue (MB) and new methylene blue (NMB), are a series of cationic thiazine dyes.33-36 Phenothiazinium photosensitizers are promising PDT agents, which exhibit excellent water solubility, high singlet oxygen quantum yields (Ф△>0.4), low dark toxicity and intense absorption in the therapeutic window (600–900 nm).33-36 However, phenothiazinium photosensitizers can be easily reduced to colorless by reductases in the body, which have negligible PDT activity.37 It has been reported phenothiazinium photosensitizers can bind with CB[8] with 2:1 mode. Fluorescence of phenothiazinium photosensitizers can be quenched due to their dimerization in the cavity of


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CB[8].31-32, 38-39 In this regard, we assumed that CB[8] may not only exhibits ability to regulate fluorescent and photodynamic activity, but also provide protective shell for the PSs inside its cavity. Herein, a CB[8] regulated aPS as a proof-of-concept for targeted cancer imaging and PDT in vivo is reported. As shown in Scheme 1, this system consists of three components: toluidine blue (PS), biotin (targeting ligand) and CB[8] (the regulator). CB[8] can bind with the biotin conjugated toluidine blue (TB-B) to form 2TB-B@CB[8] in aqueous environment, and it can also release TB-B by the competitive binding of other guest molecules including FGG tripeptide. The dimerization of TB-B in the cavity of CB[8] lead to the quenching of its fluorescence and phototoxicity. The activity of this aPS can be turned off /on by the assembly and disassembly of 2TB-B@CB[8]. CB[8] also provides excellent protection for TB-B from being reduced by enzymes, which greatly enhances the stability of TB-B in vivo. When applied for cancer treatment, 2TB-B@CB[8] is stable in blood but can be activated by intracellular triggers. With the regulation of CB[8], this system will exhibit targeted cancer imaging ability and enhanced PDT efficacy in vivo.


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Scheme 1. CB[8] regulated supramolecular aPS and its mechanism for the targeted cancer imaging and photodynamic therapy.

2. EXPERIMENTAL SECTION 2.1. Materials. Toluidine bule (TB), biotin, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), 9,10-Anthracene dipropionic acid (ADPA) salt were purchased from Sigma-Aldrich and used as received. Cucurbit[8]uril was synthesized according to a reference procedure.40 DCFH-DA was purchased from Beyotime Institute of Biotechnology (China). 2.2. Equipments. 1H NMR spectra were recorded on a Mercury VX-300 spectrometer (Varian) at 300 MHz. Fluorescent measurements were performed on a RF-5301PC spectrofluorophotometer (Shimadzu). UV/Vis measurements were carried out on a Lambda 35


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UV/VIS spectrophotometer (Perkin-Elmer). Laser flash photolysis (LFP) study was performed on LP 920 laser flash photolysis (Edinburgh Instrument). 630 nm LED light was purchased from Shenzhen LYDT technology co. (China). 660 nm laser light was purchased from Beijing StoneLaser technology co. (China). Confocal Laser Scanning Microscopy (CLSM) measurements were carried out on Nikon C1-si, BD Laser. Flow cytometry were performed by BD FACSAriaTM III. 2.3. Synthesis of TB-B. TB-B was prepared through the amidation of TB and biotin. TB (27 mg, 0.1 mmol) and biotin (48 mg, 0.2 mmol) was dissolved in 80 ml PBS buffer (10 mM, pH=5.5), followed by the addition of EDC (45.6 mg, 0.24 mmol) and NHS (27.6 mg, 0.24 mmol). The reaction was stirred at 25 oC for 24 hours. The solvent was evaporated and purified through flash chromatography (eluent: PI water and acetic acid/methanol, 1/2). After evaporation of the eluent, deep blue solid was received. 1H NMR (300 MHz, D2O): δ= 7.08 (s, 3H); 6.82 (s,2H); 6.64-6.40 (m,3H); 3.11 (m,8H); 2.85 (s,6H); 2.73 (m,4H); 1.05 (t,3H). ESI-MS calcd. for [M+]: 496.18, found: 496.19. 2.4. Fluorescent Titration. The binding constant (Kb) of TB-B and CB[8] was determined by Fluorescent spectrophotometer.41 Different amount of CB[8] (0-10 µM) was titrated to TB-B solution (4 µM) in PBS (10 mM, pH=7.4) at room temperature. TB-B was excited at 590 nm, and emissions at 647 nm were recorded. Eq.1 was used to determine the binding constant (Kb) of TB-B and CB[8]: y = 0.5R { A + B + x - ( A + B + x) − 4Bx}

(1)

Where y is the emission change at 647 nm before and after the titration of CB[8], x is the concentration of CB[8], R is a scaling factor, A is 1/Kb and B is the concentration of TB-B.


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2.5. Singlet Oxygen Measurement. The methods to measure singlet oxygen generation in solution have been previously reported.42 9, 10-Anthracene dipropionic acid (ADPA) salt was used as 1O2 indicator. The 1O2 generation ability of free TB-B and 2TB-B@CB[8] were examined upon irradiation with 630 nm LED (power density 1.3 mW/cm2) at room temperature. 1 ml solution of free TB-B (20 µM) and 2TB-B@CB[8] (10 µM) was added to ADPA solution (100 µM). After light irradiation, the UV-Vis absorption at 399 nm was recorded. The intracellular ROS generation was detected by dichlorodihydrofluorescein diacetate (DCFHDA).43 SCC-7 cells were seeded in a glass bottom dish and incubated with samples for 1h. Thereafter the cells were stained with 10 µM DCFH-DA for 20 minutes. Stained cells were washed with PBS for 3 times. The cells were incubated with fresh medium and exposed to LED light irradiation (630 nm, 30 mW/cm2, 1 minute). After irradiation, the cells were imaged by CLSM, the excitation was 488 nm, and the emission filter was 505−525 nm. 2.6. Laser Flash Photolysis (LFP) Study.

1 ml solution of TB-B (20 µM) and 2TB-

B@CB[8] (10 µM) were added in silica cuvettes and purged with air for 10 minutes, transient absorption spectra were recorded 20 ns after the excitation of 610 nm excimer laser. 2.7. Enzymatic Reduction Assay. The enzymatic reduction experiment of TB-B and 2TB-B @CB[8] was referenced from the reported method.37 0.45 µM NADH and 0.05 mg diaphorase were added in 1 ml solution of TB-B (20 µM) and 2TB-B@CB[8] (10 µM). TB-B absorption at 630 nm and 2TB-B@CB[8] at 590 nm were measured. 2.8. Components Separation and Imaging. Blood was separated to hemocyte and plasma though centrifugation. Specifically, 200µL of fresh blood from mice was collected, and 10µL of sodium citrate was added to avoid blood coagulation. Hemocyte and plasma were separated by centrifugation at 3000rpm for 10min. Hemocyte was further washed with PBS for 3 times, and


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dispersed in 100 µL of PBS. SCC-7 (squamous cell carcinoma) cells were incubated in the plate and 1×108 ~ 5×108 of them were collected. The cells were separated to nucleus, cytosol and membrane according to the Membrane and Cytoplasmic Protein Extraction Kit (Beyotime Biotech, China).44 After separation, all of them were dispersed in 1mL of PBS. The cytosol (500µL) was further added in a dialysis membrane bag (MWCO:2000), and was dialysed in PBS for 2 days (PBS was exchanged several times during the dialysis). The dialysate and the residue in the dialysis membrane bag were collected and frozen dry, and then they were dispersed in 100 µL of PBS. 100 µL of each component above was added in a 96-well plate, followed by the addition of 100 µL of 2TB-B@CB[8] (10 µM), and the samples were imaged by in vivo imaging system (Maestro, CRi Inc., Woburn, MA, USA). 2.9. Confocal Laser Scanning Microscopy (CLSM). SCC-7 cells or COS7 cells were seeded in a glass bottom dish and incubated in DMEM (1 mL) containing 10% FBS for 24 h. Then the cells were incubated with samples for 1h, and washed with PBS for 3 times after incubation. Hochest 33342 was used to stain the nucleus, 10 µg/ml of Hochest 33342 in DMEM (1 mL) was incubated with the cells for 10min, followed by PBS washing for 3 times. Biotin pretreated SCC7 cells were obtained by incubating SCC-7 cells with biotin (100 µM) for 2h. After treatments, the cells were imaged by CLSM. Fluorescence of nucleus was excited at 408 nm, and the emission filter was 420-485 nm; fluorescence of TB was excited at 637 nm, and the emission filter was over 650 nm. 2.10. Flow Cytometry. The quantitative evaluation of CLMS was performed by flow cytometry (BD FACSAriaTM III). The incubation procedure was all the same with the method in CLMS. After that, the medium was removed and the cells were washed with PBS. All the cells were digested by trypsin and collected in centrifuge tubes by centrifugating at 1200 rpm for 3


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min. The supernatant was discarded and the bottom cells were washed twice with PBS (pH 7.4). Then the suspended cells were filtrated and examined by flow cytometry. The histogram of each sample was obtained by counting 10,000 events. 2.11. In Vitro Cytotoxicity. SCC-7 cells or COS7 cells were seeded into a 96-well plate (1.5×104 cells/well) containing DMEM (200 µL). After incubation for 24 h (37 °C, 5% CO2), samples were added to the cells, after incubated with the cells for 1 hour, the cells were irradiated with LED light (630 nm, 30 mW/cm2, 1 minute) and further incubated for 24 hours. Then MTT solution (20 µL, 5 mg/mL) was added to each well and the cells were further incubated for 4 hours. Subsequently, the MTT medium was removed and DMSO (150 µL) was added to each well. The optical density (OD) was measured at 570 nm with a microplate reader (BIO-RAD 550). The cell viability was calculated as follows: Viability = (ODtreated / ODcontrol) × 100%, where ODtreated was obtained from the cells treated by the supramolecular PS system and ODcontrol was obtained from the cells without any treatments. 2.12. In Vivo Imaging and PDT Efficacy. The animal experiments were carried out in the standard A3-Lab. Xenografted SCC-7 tumour on male BALB/C nude mice model (5 weeks old) were established in mice by subcutaneously injecting a total of 1×106 SCC-7 cells. When the tumour volume reached 100 mm3, samples were injected intravenously. For in vivo imaging, the mice were imaged (Ex: 635 nm, Em: 665-695 nm ) using an in vivo imaging system (Maestro, CRi Inc., Woburn, MA, USA) at the 0, 2, 4, 8, 12, 24th hour after injection. The mice were also sacrificed 4 hours after injection, and their heart, liver, spleen, lung, kidney, muscle and tumour were collected and imaged. The tumour were also applied for cryosections. 5 µm thickness cryosections of the tumour were made and fixed with cold acetone for 10 minutes. The


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cryosections were then stained with DAPI for 10 minutes, followed by PBS washing for three times. The fluorescence of cryosections was finally imaged with CLSM. The anticancer efficacy of the samples was also examined. The mice were randomly divided into 3 groups (5 mice in each group). 4 hours after intravenous injection, the tumour site of the mice were irradiated with laser light (660 nm, 1 W/cm2, 5 minutes), and the treatment was carried out in the 1, 4, 7th day. The tumour volume and body weight of the mice were measured each day. The tumour volume was calculated as: V = W2L/2, where W is the shortest and L is the longest diameters of the tumour, respectively. At the 12th day, the mice were sacrificed, H&E staining was used for histological examination. 3. RESULTS AND DISCUSSION 3.1. Synthesis and Binding Affinity Characterization. TB-B was synthesized through the amidation reaction of TB and biotin. TB and TB-B exhibited similar UV/Vis absorption (Figure S1) and fluorescent emission (Figure S2). 2TB-B@CB[8] could be acquired by mixing TB-B and CB[8] with a molar ratio of 2:1 in water. The binding constant (Kb) of TB-B and CB[8] was determined by fluorescent titration,41 and the Kb value was determined about 2.67×107 M-1 (Figure S3). The high binding affinity host-guest interaction of TB-B and CB[8] provide a versatile strategy for the formation of distinct TB-B dimers in dilute solution. The host-guest interaction between CB[8] and the N-terminated aromatic peptides have widely been reported with a binding constant of Kb = 109 – 1011 M-2,44-46 for example, the binding constant of FGG peptide with CB[8] have been reported to be Kb=3.7±0.5×106 M-1,44, 47 which is sufficient to trigger the release of TB-B from the cavity of CB[8].


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Figure 1. A) 1H NMR spectra recorded (300 MHz, D2O, RT) for: TB-B (2 mM) and 2TBB@CB[8] (1 mM); B) UV-Vis absorption changes with the presence of CB[8] (0-10 µM) in TBB solution (20 µM), inset: image of 2TB-B@CB[8] solution (contains 20 µM of TB-B and 10 µM of CB[8]) in the left and TB-B solution (20 µM) in the right; C) Fluorescent changes with the presence of CB[8] (0-10 µM) in TB-B solution (20 µM). The formation of 2TB-B@CB[8] was confirmed through 1H NMR, UV/Vis spectroscopy and fluorescent titration. Firstly, the host-guest interaction of TB-B and CB[8] could be detected through the changes of the proton peaks by 1H NMR. As shown in Figure 1A, the signals of CB[8] were clearly detected at 5.65, 5.32 and 4.01ppm. Before and after the addition of CB[8], the signals of TB-B changed obviously. The aromatic protons of TB-B in 7.50~6.00ppm showed both downfield shifts and upfield shifts along with broaden effect, and the N-dimethyl protons


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(2.79-2.65ppm) of TB showed downfield shifts, indicating the host-guest interaction of TB-B and CB[8]. 1H−1H COSY spectra (Figure S4) helps the assignment of the peaks, and peak at 2.79 ppm does not show correlation with other peaks, proving it belong to the N-dimethyl protons of TB. ROESY NMR (Figure S5) shows correlation between TB-B and CB[8] and two-dimensional DOSY NMR (Figure S6) shows same diffuse rate between TB-B and CB[8], which further confirm the binding of TB-B and CB[8]. Secondly, UV/Vis spectroscopy was applied to examine the formation of TB-B dimers in the cavity of CB[8]. The absorption of TB monomers and dimers showed distinct absorption bands, monomers had maximum at 630 nm and dimers at 590 nm.35 UV/Vis spectroscopy observed the transformation of TB-B monomers and dimers (Figure 1B). With the addition of CB[8] (0-10 µM) to the solution of TB-B (20 µM), the absorption peak at 630 nm was weakened and finally disappeared, at the same time a new peak at 590 nm was established, proving the dimerization of TB-B inside the cavity of CB[8]. The inset images further visualized the absorption changes of TB-B (right, 20 µM) and 2TB-B@CB[8] (left, mixture of 20 µM TB-B and 10 µM CB[8]) in different colour. Thirdly, the host-guest interaction could be observed by the fluorescent titration (Figure 1C). 96.8% of the fluorescence was quenched with the appearance of 10 µM CB[8] in 20 µM TB-B. This phenomenon could be explained by the self-quenching of the closely stacked TB-B dimers in the cavity of CB[8]. 3.2. Different

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O2 Generation Ability of TB-B and 2TB-B@CB[8]. Now that the

dimerization of TB-B in the cavity of CB[8] leads to the quench of its fluorescence, we are curious about whether its photodynamic activity is also quenched or not. To answer this question, the 1O2 generation ability of TB-B and 2TB-B@CB[8] were investigated. 9, 10anthracene dipropionic acid (ADPA) was used as 1O2 indicator, which could react with 1O2 and lead to decrease in its 399-nm absorption band. The absorption decrease could be used to


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quantify 1O2 generation amount.42 As shown in Figure 2A, upon light irradiation, ADPA (100 µM) in PBS didn’t change in its absorption at 399 nm. Absorption of ADPA (100 µM) in TB-B solution (20 µM) decreased quickly, but in 2TB-B@CB[8] solution (10 µM) decreased much slower, only 12.5% of the 1O2 was generated in 6min compared with TB-B in the same condition. This observed result indicated that the 1O2 generation ability of TB-B was greatly suppressed by CB[8]. To further understand the mechanism of the suppressed 1O2 generation ability of 2TBB@CB[8], laser flash photolysis (LFP) was employed to study the transient absorption of excited states. As shown in Figure 2B, TB-B and 2TB-B@CB[8] showed distinct absorption spectra 20ns after laser light excitation. TB-B showed three major absorption peaks at 420 nm, 630 nm and 800 nm. When dyes are excited but haven’t returned to their ground states, negative peaks could be observed in LFP, these peaks are called ground state bleaching.48 The negative peak at 630 nm was assigned to ground state bleaching signal where steady-state spectral also showed absorption bands; as for the peaks at 420 and 800 nm, their decay spectra were further recorded in Figure S7, they fitted well with first-order kinetics, and exhibited similar life time (about 1.5µs). This period of life time and the position of the peaks are consistent with the triple excited state of other phenothiazine dyes, for example triple excited state of methylene blue (a similar dye with TB) also showed peaks at about 420 nm and 820 nm with a life time of 1.5~1.8 µs.36 So both of the peaks at 420 and 800 nm were assigned to TB triplet excited state (3TB*) signals. As for the condition of 2TB-B@CB[8], both of the 3TB* signals and ground state bleaching band disappeared, indicating 2TB-B@CB[8] couldn’t be excited and the energy was consumed in a much faster intermolecular electron transfer mode. To further demonstrate the proposed intermolecular electron transfer occurs only between TB pair molecules with no interaction with


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CB[8], optical properties of TB in different concentration were studied. As shown in Figure S8A, with the increase of the concentration of TB (5-300 µM), both of the absorption at 590 nm (TB dimers) 630 nm (TB monomers) increased but with different rate. In Figure S8B, ratio of absorption at 630 nm and 590nm increased in dilute solution (less than 30 µM) and decreased in high concentration, indicating TB tended to aggregate in high concentration. The aggregation of TB also quenched the fluorescence of TB, as shown in Figure S8C, fluorescent emission increased in dilute solution and then quenched along with a red shift in high concentration. In Figure S8D, fluorescence at 647 nm in different concentration (5-300 µM) showed similar transition at 30 µM as the ratio of absorption at 630 nm and 590 nm shown in Figure S8B, proving that the proposed intermolecular electron transfer occurs only between TB pair molecules with no interaction with CB[8].


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Figure 2. A) Normalized absorbance decay of ADPA (100 µM) at 399 nm in PBS (10 mM, pH=7.4), TB-B (20 µM) and 2TB-B@CB[8] (10 µM) with the irradiation of 630 nm LED light (power density 1.3mW/cm2); B) Transient absorption of TB-B (20 µM) and 2TB-B@CB[8] (10 µM) 20ns after laser light excitation. C) Mechanism of energy transfer pathways of TB and 2TB@CB[8] after light excitation. TB, 1TB* and 3TB* are ground state, singlet and triplet excited state toluidine blue. Ω1, Фf, Фnr, and ФISC are light absorption, fluorescence, nonradiative emission and intersystem crossing. From the data above, the energy transfer pathway of TB-B and 2TB-B@CB[8] could be concluded: as shown in Figure 2C, by absorbing the energy of light, ground state TB could be excited to the singlet excited state 1TB*, after intersystem crossing (ISC), the 1TB* could be transferred to triplet excited state 3TB* with a quantum yield of 0.44.35 When the 1TB* returning to the ground state, fluorescence was produced; when 3TB* returning to the ground state, the energy was transferred to the surrounding oxygen, singlet oxygen (1O2) was generated. When TB dimers formed in the cavity of CB[8], the energy of light was consumed by fast intermolecular electron transfer, the ground TB couldn’t be excited to 1TB* and 3TB*, as a result, fluorescence and ability to generate 1O2 were simultaneously quenched. 3.3. PS Releasing Behaviour. After the establishment of this supramolecular aPS, its PS delivery property would greatly influence its efficacy in PDT. It has been reported that Nterminated aromatic peptides exhibited strong binding affinity to CB[8] (Kb = 109 – 1011 M-2).4447

The competitive binding of N-terminated aromatic peptides might lead to the release of TB-B

from the cavity of CB[8]. To prove this assumption, FGG peptide was used as a model of trigger to activate 2TB-B@CB[8]. As shown in Figure 3, with the addition of FGG peptide, fluorescence of 2TB-B@CB[8] almost fully recovered in about 20 seconds. Implied that the TB-


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B can be released from the cavity of CB[8] through the competitive binding of N-terminated aromatic peptides. And the release was a fast responsive process, which was probably dependent on the diffusion rate of the triggers.

Figure 3. Fluorescent recovery of 2TB-B@CB[8] (10 µM) with the appearance of FGG peptide (100 µM) in PBS (10 mM, pH=7.4). The N-terminated aromatic peptides are likely exist in biological environment, which may activate this supramolecular photosensitizer in the body. In the following step, we examined the behavior of this supramolecular aPS in biological environment. As shown in Figure S9A, when 2TB-B@CB[8] was injected in mice, fluorescent recovery could be observed 2 hours after the injection, indicating 2TB-B@CB[8] could be activated by components in the body. To further investigate the activation mechanism of 2TB-B@CB[8], components of blood and cells were separated and their interaction to 2TB-B@CB[8] was examined. Blood was separated to hemocyte and plasma; Cells were separated to nucleus, cytosol and membrane, the cytosol was


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further separated into large molecular species (MW more than 2000) and small molecular species (MW less than 2000). The image results showed that 2TB-B@CB[8] was stable in the components in blood while could be activated in cells by small molecular species. To further investigate the possible triggers that might exist in the small molecular species, glucose, pyruvic acid, glutamine, lactic acid, collection of salts (Na+, K+, Ca2+, Mg2+, Fe3+, Zn2+), collection of 20 proteinogenic amino acids, a series of oligopeptides (GW, GY, GF, GG, FG, YG, WG, FGG) were prepared (100 µM in PBS), and their influence to 2TB-B@CB[8] was studied (Figure S9B). Only the N-terminated aromatic peptide including FG, YG, WG and FGG showed the ability to recover the fluorescence of 2TB-B@CB[8], proving the high recognition ability of CB[8], and the N-terminated aromatic peptide might be the possible trigger that existed in the cells. Unfortunately, due to the diversity and trace amount of various N-terminated aromatic peptides, to prove their existence in cells will be a challenging and systematic work, which may be studied in our following research. But the activation of 2TB-B@CB[8] in cells was undeniable. The intracellular self-activation ability of 2TB-B@CB[8] greatly improved its facility for practical uses without external inducers, which also provided potentials for disease related responses. 3.4. Intracellular Behavior of 2TB-B@CB[8]. We then examined the intracellular activation and ROS generation ability of 2TB-B@CB[8] through confocal laser scanning microscopy (CLSM) and flow cytometry analysis. SCC-7 (squamous cell carcinoma) cancerous cells were used. After incubated SCC-7 cells with TB-B (10 µM) and 2TB-B@CB[8] (5 µM) for 2 hours, fluorescence of TB was examined. As shown in Figure S10A and S10B, the cells incubated with TB-B and 2TB-B@CB[8] exhibited similar red fluorescence, flow cytometry analysis in Figure S10C further quantified these observation. The intracellular ROS generation ability of TB-B and


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2TB-B@CB[8] were examined by DCFH-DA. Non-fluorescent DCFH-DA could be oxidized by ROS to form highly fluorescent dichlorofluorescein (DCF), thus, the generated ROS could be detected.43 As shown in Figure S10D and S10E, after light irradiation, the cells incubated with TB-B and 2TB-B@CB[8] exhibited similar green fluorescence. In Figure S10F, the flow cytometry analysis also exhibited consistent result. These results indicated that 2TB-B@CB[8] was fully activated in the cells. 3.5. Cancer Cells Selectivity of 2TB-B@CB[8]. The cancer cells selectivity of 2TBB@CB[8] mediated by biotin targeting ligand was examined. SCC-7 (squamous cell carcinoma) cancerous cells were used due to their over expressing of the biotin-receptor, while COS7 (African greenmonkey kidney fibroblast cells) normal cells were used as control.49 Bright fluorescence of TB was observed in the SCC-7 cells incubated with 2TB-B@CB[8] (Figure 4A). In contrast, when SCC-7 cells were pretreated with biotin to mask the biotin acceptors, the cells showed weaker fluorescence (Figure 4B). COS7 cells incubated with 2TB-B@CB[8] also showed weaker red fluorescence (Figure 4C). Furthermore, flow cytometry analysis was used to study the mean fluorescence intensity (MFI) of TB in different samples. As shown in Figure 4D, when treated with 2TB-B@CB[8], there were 1.39-fold and 3.36-fold of MFI in SCC-7 cells compared with that in biotin pretreated SCC-7 cells and COS7 cells. The results above proved that 2TB-B@CB[8] displayed the biotin receptor-mediated endocytosis by SCC-7 cells. To verify the ligand-receptor interactions can enhance the cancer targeting ability while not caused by hydrophobic interactions or other reasons, the cancer targeting ability of 2TB-B@CB[8] and 2TB@CB[8] were compared. As shown in Figure S11A 2TB-B@CB[8] exhibited brighter red fluorescence than 2TB@CB[8] in Figure S11B. Flow cytometry analysis in Figure S11C was applied to quantitatively study the fluorescent different of the cells incubated with 2TB-


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B@CB[8] and 2TB@CB[8], 2TB-B@CB[8] exhibited 1.64 times larger MFI than 2TB@CB[8], proving the biotin ligand can enhance the cancer targeting ability of this system.

Figure 4. Confocal microscopy images showing A) SCC-7 cells incubated with 2TB-B@CB[8] (5 µM); B) Biotin (100 µM) pretreated SCC-7 cells incubated with 2TB-B@CB[8] (5 µM); C) COS7 cells incubated with 2TB-B@CB[8] (5 µM) (red=TB, blue=nucleus); D) Quantitative flow cytometry analysis of the fluorescence of 2TB-B@CB[8] (5 µM) (black line=COS7 cells control, red line=SCC-7 cells control, green line=COS7 cells incubated with 2TB-B@CB[8], blue line= biotin (100 µM) pretreated SCC-7 cells incubated with 2TB-B@CB[8] (5 µM), light blue line= SCC-7 cells incubated with 2TB-B@CB[8] (5 µM), inset: mean fluorescent intensity (MFI) of TB)


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3.6. In Vitro Cytotoxicity Assessment. The PDT efficacy of this supramolecular aPS in vitro was examined through standard MTT assay. As shown in Figure 5A, the dark/ phototoxicity of TB-B and 2TB-B@CB[8] were studied. Both TB-B and 2TB-B@CB[8] showed minimal dark toxicity, indicating that TB derivatives exhibited high safety in the dark. Acute phototoxicity of TB-B and 2TB-B@CB[8] was observed, which agreed with the result in Figure 4 that they exhibit similar ROS generation ability in cells. The targeted cancer cells killing ability was investigated by incubating 2TB-B@CB[8] with SCC-7 cells, biotin pretreated SCC-7 cells and COS7 cells. As shown in Figure 5B, SCC-7 cells treated with 2TB-B@CB[8] showed highest phototoxicity, proving the cancer cells selectivity mediated by biotin targeting ligand.

Figure 5. A) Dark toxicity and phototoxicity of TB-B and 2TB-B@CB[8] incubated with SCC-7 cells; B) Phototoxicity of COS 7 cells, biotin (100 µM) pretreated SCC-7 cells and SCC-7 cells incubated with 2TB-B @CB[8], *p < 0.05 and #p < 0.05 when SCC-7 group compared with COS 7 and SCC-7+biotin groups, by student’s t-test. 3.7. Anti-Enzymatic Reduction Assay. Phenothiazine dyes have rarely been applied for intravenous administration, this is because they are easily reduced in blood by plasma reductases,


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which greatly limits it widely application in biomedical science. To overcome this problem, the encapsulation of phenothiazine dyes in nanoparticles have been reported.50 Here the CB[8] encapsulation strategy provided a new and facile way to prevent TB from degradation in blood. The combination of diaphorase from C. kluyveri and NADH were used as a model of reduction agent to test the anti-reduction ability of 2TB-B@CB[8].37 As shown in Figure 6 and Figure S12, TB-B and 2TB-B@CB[8] were added to the solution of reduction agent, their absorption at 630 nm and 590 nm were recorded versus time respectively. Free TB-B was almost totally reduced to colorless within 30 minutes, while about 95% of the TB-B in CB[8] was not reduced, proving the outstanding protection ability of CB[8] to TB-B.

Figure 6. Absorbance decay of TB-B at 630 nm and 2TB-B@CB[8] at 590 nm with the appearance of NADH (0.45 µM) and diaphorase (0.05 mg) in PBS (10 mM, pH=7.4). 3.8. In Vivo Imaging and Antitumour Efficacy. The feasibility of 2TB-B@CB[8] in vivo was investigated through the SCC-7 tumour-bearing nude mice model. After intravenously injecting of TB-B and 2TB-B@CB[8] (2 mg/kg), in vivo biodistribution and retention were traced via small animal imaging system. As shown in Figure 7A, after the injection of 2TB-


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B@CB[8], fluorescent signal could be observed, due to the weak fluorescence of TB and poor tissue penetration ability of the fluorescence from TB, only limited signals could be observed. But the activation and accumulation of 2TB-B@CB[8] in tumour was obvious. Strongest fluorescent signal was observed 4h after injection, then the fluorescence was gradually decreased and almost disappeared after 24h. In contrast, without the protection of CB[8], TB-B showed less tumour accumulation ability and much faster elimination rate in the body. The distribution of TB-B and 2TB-B@CB[8] in organs was also carried out 4h after injection, the mice were sacrificed and their blood(a), lung(b), muscle(c), liver(d), spleen(e), kidney(f), heart(g) and tumour(h) were collected and imaged. As shown in Figure 7A, although TB-B and 2TBB@CB[8] inevitably accumulation in metabolic organs, liver and kidney, their accumulation in tumour tissue was in great contrast, 2TB-B@CB[8] exhibited better tumour accumulation ability than TB-B. To further verify the better accumulation of 2TB-B@CB[8] in tumour, cryosectioned tumour tissues injected with TB-B and 2TB-B@CB[8] were studied through CLSM observation. As shown in Figure 7B, brighter red fluorescence could be observed in 2TB-B@CB[8] than TBB. These result could be explained as follow: 2TB-B@CB[8] was stable in blood circulation and could be activated in cells, when it appeared in blood, CB[8] protected TB-B from being reduced by the enzymes in blood, as a result, longer retention time and then better accumulation in tumour could be achieved by 2TB-B@CB[8]. The antitumour efficacy of 2TB-B@CB[8] was evaluated with the irradiation of laser light (660 nm, 1W/cm2, 5 minutes), and the treatment was carried out at the 1, 4, 7th day. From the tumour volume results in Figure 7C, 2TB-B@CB[8] showed better tumour growth inhibition ability than TB-B. Similar results could be observed in the picture of the mice after therapy (Figure S13). The low systemic toxicity of 2TB-B@CB[8] was confirmed through the unobvious


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body weight changes (Figure S14), and all of the mice were survived at the end of the treatment. Above result was further confirmed by hematoxylin and eosin (H&E) staining in Figure 7D, the damages in tumour tissue were in great comparison. When the mice were treated with 2TBB@CB[8], most of the tumour cells were killed, while the mice treated with TB-B showed limited apoptosis/necrosis in tumour tissue, and for the PBS group, most of the tumour cells were survived. But no obvious damages were found in the slices of other organs.


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Figure 7. In vivo imaging and PDT efficacy of 2TB-B@CB[8]. A) In vivo imaging of TB-B and 2TB-B@CB[8] (2 mg/kg) after intravenous administration through tail vein (white circle: tumour region), and organs distribution of TB-B and 2TB-B@CB[8] in blood (a), lung (b), muscle (c), liver (d), spleen (e), kidney (f), heart (g) and tumour (h) 4 hours after injection; B) Confocal microscopy images of crysectioned tumour tissues injected with TB-B and 2TB-B@CB[8] (red=TB, blue=nucleus); C) Relative tumour volume change during the treatment (black arrows: the treatment was carried out in the 1, 4, 7th day). * p < 0.05 and # p < 0.05 when 2TB-B@CB[8] group compared with PBS and TB-B groups, by student’s t-test; D) H&E staining images of the organs at the 12th day after treatment. 4. CONCLUSIONS In summary, we have successfully developed a supramolecular aPS system for targeted cancer imaging and PDT in vivo. Reversible control of fluorescence and photodynamic activity were achieved by the assembly and disassembly of 2TB-B@CB[8], and the mechanism of this control was proved by LFP. This system combines a series of key functions such as targeting, protection, transport, delivery, imaging and therapy into a single supramolecular complex. In vivo results showed that 2TB-B@CB[8] improved the stability of TB-B for intravenous administration, which presented longer retention in the body, better accumulation in tumour and enhanced cancer ablation ability. This supramolecular regulation strategy provides a facile, fast responsive, reversible and economic way to fabricate aPS. Many fluorescent dyes or dye derivatives have been reported to have host-guest interaction with macrocycles, and the present supramolecular systems provides potentials to regulate many other fluorescent dyes and photosensitizers in biomedical science.


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ASSOCIATED CONTENT Supporting Information. Experimental details can be found in the SI. This SI material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors * Corresponding Authors. [email protected] (Y. X. Sun), [email protected] (X. Z. Zhang). Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51473126, 51233003 and 51533006) and the Fundamental Research Funds for the Central Universities. REFERENCES (1)

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Cucurbit[8]uril Regulated Activatable Supramolecular Photosensitizer

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