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Sep 13, 2017 - suppressed by CPZ, which is known to block the clathrin- mediated endocytosis. In contrast, cellular uptake of OPVBT was totally inhibi...
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Oligo(p‑phenylenevinylene) Derivative-Incorporated and EnzymeResponsive Hybrid Hydrogel for Tumor Cell-Specific Imaging and Activatable Photodynamic Therapy Meng Li, Ping He, Shengliang Li, Xiaoyu Wang, Libing Liu,* Fengting Lv, and Shu Wang* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China S Supporting Information *

ABSTRACT: Tumor-cell targeted and subcellular organelles-specific photosensitizer is imperative for high-efficiency photodynamic therapy (PDT). Herein, we designed and synthesized a new conjugated oligomer, oligo(pphenylenevinylene-co-benzothiazole) (OPVBT), which could be used as photosensitizer to generate reactive oxygen species (ROS) upon light illumination. Advantageously, OPVBT was found to specifically accumulate in mitochondria, which is beneficial for effective subcellular organelles-targeted PDT. A biocompatible and biodegradable hydrogel was constructed based on Michael-type addition between cysteine-terminated matrix metalloproteinase (MMP)-cleavable oligopeptides and 4-arm vinyl sulfone-terminated poly(ethylene glycol) (PEG-VS). By functionalizing hydrogel with OPVBT and cell adhesion peptide motif RGD, cell attachment and spreading on the hydrogel were easily realized. More importantly, because of the MMPsensitivity of the hydrogel, tumor cells could be specifically lighted up by OPVBT liberated from the hydrogel and the growth of tumor cells are further restrained by ROS generated from OPVBT upon light illumination. The hybrid hydrogel provides a new platform for tumor-specific imaging and killing, which shows good potential for constructing targeted PDT system with reduced side effects. KEYWORDS: conjugated oligomer, responsive hydrogel, cell imaging, photodynamic therapy

1. INTRODUCTION Photodynamic therapy (PDT) is a medical treatment that involves a photosensitizer and a light source. Upon light activation, the photosensitizer could sensitize surrounding oxygen molecules to generate reactive oxygen species (ROS) that destroy nearby cells. As a minimally invasive therapeutic strategy, PDT is widely used for oncotherapy, especially for skin cancer treatment.1−3 Despite achievements have been made in tumor treatment, considerable attention has been paid to develop new PDT systems with improved therapeutic efficiency and reduced side effects.4,5 Because ROSs have a short half-life and small effective radius of action, it is expected that PDT efficiency would be improved if the photosensitizer is targeted to tumor cells and delivered into specific subcellular organelles.6−8 The deficiency of side effects for normal cells has always existed in cancer therapeutic methods. Considerable reports have confirmed that many cancer cells have elevated matrix metalloproteinase (MMP) level, and MMP is closely implicated in tumor growth, invasion, and metastasis.12,13 Thus, many MMP-related anticancer strategies were proposed, especially MMP-sensitive materials for stimuli-responsive drug release to reduce side effect for normal cells.14−17 As we all know, mitochondria are crucial subcellular organelles and play a vital © XXXX American Chemical Society

role not only in energy production but also in cellular signaling, which is related to cell growth, differentiation, and apoptosis. Therefore, many efforts have been taken to develop mitochondria-targeted photosensitizers for enhanced PDT.9−11 Among stimuli-sensitive materials, hydrogels have become especially attractive. Hydrogels are three-dimensional crosslinked polymers which are characterized as high porosity, permeability for nutrients and metabolites, and have been extensively applied as cell culture matrix and tissue engineering material.18−20 Recently, stimuli-responsive hydrogels have drawn increasing interest as drug carrier in tumor therapy due to the facile encapsulation of bioactive substrates by simply blending them in precursor solution.21−23 However, hydrogels, which could be used for tumor cell-specific mitochondria imaging and photodynamic therapy simultaneously, have been rarely reported. In this work, we synthesized a new cationic oligo(p-phenylenevinylene-co-benzothiazole) (OPVBT) which happens to accumulate in mitochondria. OPVBT is capable of generating vast ROS upon light irradiation and shows Special Issue: Biomaterials Science and Engineering in China Received: August 23, 2017 Accepted: September 13, 2017 Published: September 13, 2017 A

DOI: 10.1021/acsbiomaterials.7b00610 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

40 mL of degassed toluene was added tetrakis(triphenylphosphine) palladium (143 mg, 0.124 mmol) under Ar atmosphere. The mixture was stirred at 90 °C for 5 h. The reaction was quenched by addition of 40 mL of 1 M potassium fluoride aqueous solution. The organic layer was dried over anhydrous MgSO4. After the solvent was removed in vacuum, the resulting residue was purified by silica gel chromatography with petroleum ether/dichloromethane (1:1) as eluent to afford faint yellow solid (713 mg, 85%). 1H NMR (400 MHz, Acetone) δ 8.60 (s, 1H), 7.56 (d, 2H), 7.47 (d, 2H), 7.39 (s, 1H), 7.27 (d, 2H), 7.17 (s, 1H), 7.04 (dd, 1H), 5.82 (d, 1H), 5.21 (d, 1H), 4.07 (t, 4H), 3.51 (td, 4H), 1.86 (td, 8H), 1.55 (dd, 8H), 1.49 (s, 9H). 13C NMR (125 MHz, Acetone) δ 153.61, 151.68, 140.12, 133.06, 132.35, 129.52, 128.19, 127.73, 127.28, 122.23, 119.19, 114.18, 111.38, 111.12, 80.07, 69.82, 69.59, 34.70, 34.34, 33.59, 33.57, 30.89, 28.59, 28.53, 26.21, 26.10, 25.96, 25.86. HRMS (MALDI) m/z: [M]+ calcd. 677.17099, found 677.17055. 2.5. Synthesis of Compound 7. To a solution of compound 6 (265 mg, 0.39 mmol) and compound 2 (185 mg, 0.59 mmol) in 25 mL of toluene were added tri-o-tolylphosphine (48 mg, 0.16 mmol) and tributylamine (187 μL, 0.78 mmol). The mixture was degassed and palladium(II) acetate (9 mg, 0.04 mmol) was added. The mixture was stirred at 110 °C for 24 h. After solvent was removed under vacuum and the resulting residue was purified by silica gel chromatography with petroleum ether/dichloromethane (1:3) as eluent to afford yellow solid (120 mg, 35%). 1H NMR (400 MHz, CDCl3) δ 8.10 (d, 1H), 7.63 (d, 2H), 7.47 (d, 2H), 7.39 (d, 2H), 7.36 (d, 2H), 7.33 (d, 1H), 7.17 (s, 1H), 7.12 (s, 1H), 6.53 (s, 1H), 4.09 (dd, 4H), 4.06 (s, 3H), 3.43 (dd, 4H), 1.92 (d, 8H), 1.69−1.55 (m, 8H), 1.53 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 157.23, 152.73, 151.43, 151.04, 147.73, 138.00, 134.67, 134.22, 132.82, 129.03, 128.36, 127.79, 127.40, 125.93, 124.92, 121.99, 121.39, 120.78, 118.76, 113.42, 113.10, 110.99, 110.08, 80.83, 69.69, 69.09, 56.91, 33.95, 33.57, 33.54, 32.84, 30.47, 30.42, 29.58, 29.48, 28.50, 28.15, 28.10, 25.73, 25.67, 25.49, 25.43. HRMS (MALDI) m/z: [M]+ calcd. 865.175418, found 865.176595. 2.6. Synthesis of OPVBT. To a solution of compound 7 (8 mg, 9 μmol) in 2 mL of THF, 1 mL of 1 M trimethylamine in THF was added. After reacted at 40 °C for 12 h, 3 mL of DMF was added to dissolve the precipitate. The mixture was reacted at 40 °C for another 24 h. The solvent was removed under vacuum and 1 mL of DMF was added. The mixture was poured to 10 mL of petroleum ether/ dichloromethane (1:1) and yellow precipitate was obtained (7 mg, 78%). 1H NMR (400 MHz, DMF) δ 9.51 (s, 1H), 8.29 (d, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 7.75−7.68 (m, 1H), 7.65 (d, 2H), 7.57 (d, 2H), 7.52 (s, 2H), 7.49 (s, 2H), 4.23 (dt, 4H), 4.16 (s, 3H), 3.64−3.49 (m, 4H), 3.35 (s, 18H), 2.10−1.79 (m, 8H), 1.79−1.58 (m, 8H), 1.51 (s, 9H). 13C NMR (126 MHz, DMF) δ 157.98, 153.44, 151.71, 151.26, 140.13, 134.40, 132.24, 129.79, 128.40, 127.88, 127.35, 125.66, 125.45, 121.97, 121.31, 120.36, 118.62, 114.44, 113.90, 111.28, 110.53, 100.27, 79.54, 69.53, 69.17, 67.60, 66.35, 57.16, 28.05, 26.45, 26.36, 26.15, 26.12, 25.64, 22.98. HRMS (ESI) m/z: [M-2Br]2+ calcd. 412.742597, found 412.742405. 2.7. Surface Tension Measurement. The surface tension measurements of OPVBT in 1 × phosphate buffer solution (PBS) were performed with a Pt/Ir plate method on a DCAT21 tensiometer (Dataphysics Co., Germany) at 25.00 ± 0.01 °C. The tensiometer was calibrated by testing pure water before each set of measurements. The tests were repeated three times. 2.8. Reactive Oxygen Species (ROS) Measurement. To 1 mL of 40 μM activated DCFH in 1× phosphate buffer solution (PBS), was added OPVBT with the final concentration of 1 μM, 5 μM, 10 μM, or 20 μM. The solution was irradiated by white light at the power of 2 mW cm−2 or 0.5 mW cm−2. Nonfluorescent DCFH could be converted into DCF with intense green fluorescence in the presence of ROS. The fluorescent intensity at 528 nm was recorded using a microplate reader with the excitation wavelength of 485 nm after different durations of irradiation time. 2.9. Cell Culture. MCF-7, MDA-MB-231 human breast cancer cells and 293T normal cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum

significant phototoxicity as a new photosensitizer. To obtain a targeted PDT system, an MMP-sensitive PEG-peptide hydrogel was constructed to improve tumor selectivity and reduce side effects. The hydrogel was incorporated with OPVBT and cell adhesion peptide motif RGD to jointly promote cell adhesion. Due to the elevated MMP level in tumor cells, the hydrogel containing a cleavable substrate for MMP could be gradually degraded to locally release the incorporated OPVBT that was endocytosed by tumor cells. Therefore, the tumor cells could be specifically lighted up by OPVBT on the hydrogel and further killed by ROS generated from OPVBT upon light irradiation.

2. EXPERIMENTAL SECTION 2.1. Materials and Instruments. Chemicals were purchased from commercial suppliers (Acros, Sigma-Aldrich, Alfa-Aesar et al.) and used as received without further treatment unless otherwise noted. Four-arm PEG (4-arm-PEG-VS, 20 kDa) was purchased from JenKem Technology Co., Ltd. (Beijing, China). Oligopeptide (CRD-VPMS↓ MRGG-DRC) and c(RGDfC) were obtained from ChinaPeptides Co., Ltd. (Shanghai, China). Mito-Tracker Red-FM was obtained from Invitrogen/Thermo Fisher Scientific Inc. (USA). 293T, MCF-7, and MDA-MB-231 cells were purchased from cell culture center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Twenty-four well glass bottom plate (black plate with lid, 0.17 ± 0.005 mm, P24−1.5H−N) was purchased from in vitro scientific (Cellvis, USA). The 1H NMR and 13C NMR spectra were recorded on Bruker Avance 400 and Bruker Avance III 400 HD. High resolution mass spectra (HRMS) were recorded on a Bruker 9.4T Solarix FT-ICR-MS (Bruker, Germany). UV−vis absorption spectra were conducted on a JASCO V-550 spectrophotometer (JASCO, Japan). Fluorescence emission spectra were taken on a Hitachi F-4500 fluorometer equipped with xenon lamp excitation source (Hitachi, Japan). Confocal laser scanning microscopy (CLSM) images were taken on Olympus FV 1200-BX61 (Olympus, Japan). White light source (400−800 nm) with a xenon lamp (CXE-350) was provided by Beijing OPT Photoelectric Technology Co., Ltd. 2.2. Synthesis of Compound 2. To a solution of 2-cyano-6methoxybenzothiazole (compound 1, 910 mg, 4.8 mmol) in 30 mL of dichloromethane and 30 mL of acetic acid was added Niodosuccinimide (3.25 g, 14.4 mmol). The mixture was stirred at room temperature for 2 days. The mixture was washed with 10% sodium thiosulfate aqueous solution and then washed twice with water. The solvent was removed under vacuum and the residue was purified by silica gel chromatography with petroleum ether/dichloromethane (1:1) as eluent to afford white solid (1.51 g, 98%). 1H NMR (400 MHz, CDCl3) δ 8.15 (d, 1H), 7.18 (d, 1H), 4.03 (s, 3H), 1.52 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 159.28, 145.94, 144.66, 132.86, 126.01, 113.21, 112.20, 77.48, 77.16, 76.84. HRMS (MALDI) m/z: [M + H]+ calcd. 316.92400, found 316.92410. 2.3. Synthesis of Compound 5. To a solution of compound 3 (1.1 g, 5 mmol) and compound 4 (9.6 g, 15 mmol) in 120 mL of toluene, were added tri-o-tolylphosphine (608 mg, 2 mmol) and tributylamine (2.4 mL, 10 mmol). The mixture was degassed and palladium(II) acetate (112 mg, 0.5 mmol) was added. The mixture was stirred at 110 °C for 24 h. After solvent was removed under vacuum, the residue was eluted with petroleum ether/dichloromethane (5:1) through silica gel chromatography to recycle the excess compound 4 (4.6 g, recycle yield 72%) and then eluted with petroleum ether/ dichloromethane (1:1) to afford white solid product (2.83 g, 77%). 1H NMR (400 MHz, Acetone) δ 8.49 (s, 1H), 7.58 (d, 2H), 7.48 (d, 2H), 7.38 (d, 1H), 7.34 (s, 1H), 7.30 (s, 1H), 7.20 (s, 1H), 4.08 (dt, 4H), 3.52 (td, 4H), 1.87 (qt, 8H), 1.67−1.51 (m, 8H), 1.49 (s, 9H). 13C NMR (101 MHz, Acetone) δ 153.62, 151.82, 150.74, 140.32, 132.81, 130.27, 127.97, 127.83, 121.65, 119.20, 118.65, 112.28, 111.55, 80.11, 70.37, 70.13, 34.69, 34.70, 33.58, 28.53, 26.11, 25.97. HRMS (MALDI) m/z: [M]+ calcd. 729.06585, found 729.06632. 2.4. Synthesis of Compound 6. To a solution of compound 5 (905 mg, 1.24 mmol) and tributylvinylstannane (1.6 mL, 5.5 mmol) in B

DOI: 10.1021/acsbiomaterials.7b00610 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Scheme 1. Synthetic Route of OPVBT

(FBS), 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin. All cell lines were cultured at 37 °C in humidified incubator with 5% CO2 and 95% air. 2.10. Cell Imaging and Colocalization Assays. MCF-7 cells were seeded in 20 mm glass bottom cell culture dishes at a density of 8 × 104 cells per dish and cultured to attach for 12 h. Cells were washed with PBS and OPVBT in culture medium was added to the adherent cells. After incubated for 4 h, cells were treated with Mito-Tracker Red-FM at a final concentration of 250 nM in serum-free medium for 20 min. Cells were rinsed with PBS and observed immediately by confocal laser scanning microscopy. For OPVBT, the excitation laser was 405 nm, for Mito-Tracker Red-FM, the excitation laser was 559 nm. 2.11. Cellular Uptake Experiment. MCF-7 cells were seeded in 20 mm glass bottom cell culture dishes at a density of 8 × 104 cells per dish and cultured to attach for 12 h. As shown in Table S1, cells were then pretreated with serum free medium at 4 °C or serum free medium containing dynasore (80 μM), chlorpromazine (10 μg mL−1), and genistein (50 μg mL−1) at 37 °C for 1 h, respectively. Then, 5 μM OPV in the above condition medium was added into each dish and cultured for another 4 h. The cells were washed twice with PBS before CLSM imaging. 2.12. In Vitro Cytotoxicity of OPVBT. The cells were seeded in 96-well plates at a density of 4 × 103 cells per well. After 12-h incubation, the media were replaced by various concentrations of OPVBT in culture medium. For the dark-toxicity groups, cells were allowed to grow for another 48 h. For the phototoxicity groups, cells were cultured for 12 h, after which the cells were irradiated with white light at a light density of 1 mW cm−2 or 2 mW cm−2 for 30 min followed by incubation for 24 h. Then the culture medium was replaced by MTT solution (0.5 mg mL−1, 100 μL per well). After 4 h

of incubation, the medium was removed and DMSO (100 μL per well) was added to dissolve the precipitate assisted by shaking. The absorbance values of each well at 570 nm were measured by a microplate reader. Cells without OPVBT treatment were used as control. 2.13. Matrix Mtalloproteinase (MMP) Activity Assay. The MMP activity assay kit was purchased from Abcam (Cat No. ab112146) and used according to the instructions. MCF-7 cells were seeded into triplicate wells of 6-well plates at the density of 2 × 105 cells per well and allowed to attach overnight. Then the cells were starved with serum-free culture medium for another 24 h. MMP activity in the conditioned medium was tested. In brief, 25 μL of medium was added to 25 μL of 2 mM 4-aminophenylmercuric acetate (APMA) working solution and incubated at room temperature for 15 min to activate zymogen. After that, 50 μL of the MMP green substrate working solution was added and incubated at room temperature for 4 h. The MMP green substrate supplied in the kit is nonfluorescent due to a linked quencher. After cleaved by MMP, the green fluorescence is recovered. The fluorescent intensity was recorded using a microplate reader with a filter set of Ex/Em = 485 nm/528 nm. 2.14. Preparation of Hydrogel. (a) Stock solution of OPVBT, oligopeptides, PEG-VS, and RGD: 20 mM OPVBT in DMF: 12.7 mg of OPVBT dissolved in 645 μL of DMF. Twenty mM oligopeptide aqueous solution: 63.3 mg of oligopeptides dissolved in 2 mL of water. 10% w/v PEG-VS aqueous solution: 500 mg of PEG-VS dissolved in 5 mL of water. Five mM RGD aqueous solution: 20 mg of c(RGDfC) dissolved in 6.92 mL of water. (b) 2.8% hydrogel preparation ([OPVBT] = 200 μM, [RGD] = 200 μM): 2 μL of 20 mM OPVBT was mixed with 48 μL of CH3CN and 25 μL of 20 mM oligopeptides aqueous solution. The mixture was allowed to react at 37 °C for 3 h. C

DOI: 10.1021/acsbiomaterials.7b00610 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Then 75 μL of 0.8 M TEA buffer solution (pH 7.9), 8 μL of 5 mM RGD aqueous solution, and 25 μL of 10% w/v PEG aqueous solution were added. The solution of precursors was allowed to form hydrogel at 37 °C overnight. 2.15. Cell Attachment and Spreading on the Hydrogel. The solution of precursors with different concentrations of OPVBT and RGD were transferred into glass-bottom 24-well plate with 120 μL per well ((1) [RGD] = 0, [OPVBT] = 0; (2) [RGD] = 50 μM, [OPVBT] = 0; (3) [RGD] = 0, [OPVBT] = 100 μM; (4) [RGD] = 50 μM, [OPVBT] = 100 μM). Gelation reaction was carried out overnight to achieve utmost cross-linking efficiency. Then, the hydrogel was immersed with 2 mL of PBS, and washed with PBS for 5 times. PBS was replaced with culture medium and immersed overnight after which cells were seeded on the hydrogel at a density of 1 × 105 cells per well. After incubated for different time intervals, cells were imaged with CLSM. 2.16. Cell Imaging by the Hydrogel. The solution of precursors was transferred into glass-bottom 24-well plate with 120 μL per well ([OPVBT] = 200 μM, [RGD] = 200 μM). Gelation reaction was carried out overnight to achieve utmost cross-linking efficiency. Then, the hydrogel was immersed with 2 mL of PBS, and washed with PBS for 5 times. PBS was replaced by culture medium and immersed overnight after which cells were seeded on the hydrogel at a density of 1 × 105 cells per well. After incubated for different time intervals, cells were imaged with CLSM. The excitation laser of OPVBT was 405 nm. 2.17. In Vitro Cytotoxicity of Hydrogel. The solution of precursors was transferred into glass-bottom 96-well plate with 20 μL per well ([RGD] = 200 μM, [OPVBT] = 0, 50, 100, 150, or 200 μM). Gelation reaction was carried out overnight to achieve utmost crosslinking efficiency. Hydrogel was washed with PBS on Wellwash instrument for 10 times. PBS was replaced by culture medium and immersed overnight after which cells were seeded on the hydrogel (MCF-7 cells were added at a density of 5 × 105 cells per well, MDAMB-231 and 293T cells were added at a density of 8 × 105 cells per well). For the dark-toxicity groups, cells were allowed to grow for 48 h. For the phototoxicity groups, cells were cultured for 24 h, after which the cells were irradiated with white light at a density of 1 mW cm−2 or 2 mW cm−2 for 30 min followed by incubating for another 24 h. Then the culture medium was replaced by MTT solution (0.5 mg mL−1, 100 μL per well). After 4-h incubation, the medium was removed and DMSO (100 μL per well) was added to dissolve the precipitate with shaking. The absorbance values of each well at 570 nm were measured by a microplate reader (BioTek Synergy HT).

Figure 1. (a) Normalized UV−vis absorption and fluorescence emission spectra of OPVBT in water. [OPVBT] = 10 μM. (b) Fluorescence emission intensity at 528 nm of DCFH in phosphate buffer solution with or without OPVBT as a function of light irradiation time. The power density of white light is 2 mW cm−2. [DCFH] = 40 μM, [OPVBT] = 1 μM.

electron-withdraw ability of CBT group on the conjugated backbone, the fluorescence quantum yield of OPVBT is low (∼2%); however it still has a bright signal under the confocal laser scanning microscopy (see the cell imaging results below). The ROS generation ability of OPVBT upon white light irradiation was investigated utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as a probe. DCFH-DA could be activated into 2′,7′-dichlorodihydrofluorescein (DCFH) under alkaline conditions and further converted into highly fluorescent 2′,7′-dichlorofluorescein (DCF) upon oxidation by ROS. As shown in Figure 1b, while DCFH (40 μM) solution was irradiated under white light (2 mW cm−2) in the presence of OPVBT (1 μM), fluorescent intensity at 528 nm increased linearly over time. By contrast, for DCFH solution without OPVBT, fluorescent intensity at 528 nm barely changed. These results confirm that massive ROS are generated from OPVBT upon white light irradiation. Considering the amphiphilic property of OPVBT, the surface tension (SFT) measurement was utilized to test the critical micellar concentration (CMC) in 1× phosphate buffer solution (PBS). As shown in Figure S1a, the CMC measured from the surface tension curve is 2 μM, indicating that OPVBT aggregated in PBS when the concentration is over 2 μM. Thus, the ROS generation ability of OPVBT was further tested with a final concentration of 5, 10, and 20 μM under the light density of 0.5 mW cm−2. As shown in Figure S1b, OPVBT shows linearly increased ROS

3. RESULTS AND DISCUSSION The synthetic route of OPVBT is illustrated in Scheme 1. Compound 2 was prepared through the reaction of compound 1 with N-iodosuccinimide in 98% yield. Compound 5 was obtained by the coupling reaction of compound 3 with compound 4 in a yield of 77%. Compound 5 reacted with tributylvinylstannane to yield compound 6 (yield: 85%) followed by reaction with compound 2 to afford compound 7 in a yield of 35%. OPVBT was finally obtained by treatment of compound 7 with trimethylamine in 78% yield. OPVBT possesses hydrophobic backbone and positively charged side chains, thus it is envisaged that OPVBT could easily bind to cells through hydrophobic and electrostatic interactions. Meanwhile, OPVBT was functionalized with a reactive group, 2-cyano-6-methoxybenzothiazole (CBT),24−26 which has been widely reported to react with N-terminal cysteine smoothly in aqueous solution, and thus OPVBT could be further covalently linked to hydrogels. The photophysical properties of OPVBT were tested in water. As shown in Figure 1a, OPVBT showed maximum absorption peaks at 331 and 392 nm and a maximum emission at 485 nm. The fluorescence quantum yield of OPVBT was tested in water using quinine sulfate as a standard. Due to the D

DOI: 10.1021/acsbiomaterials.7b00610 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 2. (a) CLSM images and (b) normalized fluorescence intensities of MCF-7 cells treated with OPVBT along with different endocytosis inhibition conditions. [OPVBT] = 5 μM. (c) The CLSM images of MCF-7 cells stained with OPVBT and Mito-Tracker Red-FM and the colocalization scatter plot. [OPVBT] = 5 μM, [Mito-Tracker] = 250 nM. The false colors of OPVBT and Mito-Tracker Red-FM are green and red, respectively. The merged color of OPVBT and Mito-Tracker Red-FM is yellow.

Figure 3. Cell viabilities of 293T, MCF-7, and MDA-MB-231 cells treated with OPVBT using MTT assay under dark or white light irradiation at a light dose of 1.8 J cm−2 or 3.6 J cm−2.

amount until the DCFH is completely consumed, suggesting that the aggregation of OPVBT in PBS does not affect the ROS generation. To investigate cell imaging ability of OPVBT, human breast carcinoma cells MCF-7 were used as representative. As shown in Figure S2, OPVBT could enter into cells within 30 min and was widely distributed in the whole cytoplasm. With the incubation time extended, the amount of OPVBT in cells increased and maintained wide distribution. To further confirm the endocytosis mechanism of OPVBT, endocytosis inhibition experiments were conducted. MCF-7 cells were treated with OPVBT along with different endocytosis inhibition conditions for 4 h, including low-temperature (4 °C), chlorpromazine (CPZ), dynasore, and genistein, respectively (Table S1). Then cells were imaged using confocal laser scanning microscopy (CLSM) (Figure 2a), and the fluorescent intensity of OPVBT in each image was calculated for quantitative analysis (Figure 2b). The results indicated that the uptake of OPVBT was

strongly inhibited at low-temperature, which reveals that OPVBT internalization process is energy-dependent endocytosis. In addition, the internalization of OPVBT was scarcely suppressed by CPZ, which is known to block the clathrinmediated endocytosis. In contrast, cellular uptake of OPVBT was totally inhibited in the presence of dynasore, which is known to inhibit dynamin function essential for the formation of a pinched-off vesicle and is necessary for both clathrin- and caveolae-mediated endocytosis. Also, OPVBT uptake was suppressed by genistein which is capable of blocking caveolae-mediated route. Therefore, OPVBT internalization process is mainly caveolae-mediated endocytosis which can bypass the lysosome trafficking and promote the subcellular targeting.27,28 To determine the subcellular localization of OPVBT, we applied a mitochondria-located dye (Mito-Tracker Red-FM) for colocalization. As shown in Figure 2c, OPVBT displayed a typical mitochondrial localization pattern and was well-merged with Mito-Tracker Red-FM with a Pearson’s E

DOI: 10.1021/acsbiomaterials.7b00610 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 4. (a) Illustration of MMP-sensitive hydrogel incorporated with OPVBT and RGD for tumor cell-specific imaging and photodynamic therapy. (b) Photographs of precursors in buffer solution and the hydrogel after incubation at 37 °C for 30 min.

correlation coefficient of 0.91. As previously reported, mitochondrial membrane potential is about −180 mV, which is much higher than that of other subcellular organelles (−40 to −80 mV), thus cationic OPVBT could be easily attracted by mitochondria via electrostatic interactions.29 Considering the effective ROS generation capability and the mitochondria-specific targeting ability of OPVBT, we speculated that OPVBT might have strong phototoxicity toward cells. Therefore, cytotoxicity of OPVBT under dark or white light irradiation was tested toward three cell types, including human embryonic kidney cells (293T) and human breast carcinoma cells (MCF-7 and MDA-MB-231). As shown in Figure 3, for the dark groups, OPVBT exhibited low cytotoxicity toward all tested cell types even at a high concentration of 20 μM. On the contrary, OPVBT showed much higher cytotoxicity toward all tested cells under white light irradiation. Viabilities of all the three kinds of cells were lower than 10% when cells were exposed to 20 μM OPVBT and a light dose of 3.6 J cm−2. These results reveal that OPVBT shows great potential as a new mitochondria-located photosensitizer in PDT application. Taking advantage of excellent mitochondria-targeted phototoxicity of OPVBT, a MMP-sensitive hydrogel system was constructed. As shown in Figure 4a, the hybrid hydrogel was prepared by Michael-type addition of cysteine-terminated oligopeptides onto 4-arm vinyl sulfone-terminated poly(ethylene glycol) (PEG-VS) at physiological temperature. PEG chains were chosen due to the inherent biocompatibility

and resistance to nonspecific adsorption. The tailor-made oligopeptide was utilized to realize biological functionality. A MMP cleavable sequence (CRD-VPMS↓MRGG-DRC, the symbol ↓ means the cleavage site) is selected, so that the hydrogel is sensitive to proteolytic degradation by MMP secreted from tumor cells. Since the CBT unit on OPVBT is capable of reacting with N-terminated cysteine of oligopeptide while leaving C-terminated cysteine for gelation, OPVBT could be covalently cross-linked into the hydrogel. To obtain cellresponsive hydrogel, we also incorporated cysteine-functionalized cyclopeptide c(RGDfC) (abbreviated as RGD below) into the hydrogel network by Michael-type addition reaction to benefit cell adhesion. As shown in Figure 4b, the gelation could occur within 30 min at 37 °C, as a matter of fact, the gelation precursors were mixed and incubated overnight at 37 °C to achieve utmost cross-linking efficiency. To manifest the biocompatibility of the hybrid hydrogel, we investigated the cell attachment and spreading on the hydrogel using MCF-7 cells as representative. The hydrogel precursors with different concentrations of OPVBT and RGD were allowed to gelate on the cell culture plate, and then MCF-7 cell suspension was seeded onto the hydrogel and incubated for 24 h. As shown in Figure 5a, when the hydrogel contains neither OPVBT nor RGD, cells could not adhere to the hydrogel. As for hydrogel contains only OPVBT or RGD alone, cells could adhere to the hydrogel but not as spreading as on the hydrogel consisting of both OPVBT and RGD. The cells in each image were counted by choosing three visual fields randomly. Due to F

DOI: 10.1021/acsbiomaterials.7b00610 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 5. (a) Cell attachment and spreading on the hydrogel containing different concentrations of OPVBT and RGD. The numbers on the images stand for cell numbers counted by choosing three visual fields randomly. (b) MMP activities of different cells determined using an MMP activity assay kit. (c) CLSM images of 293T, MCF-7, and MDA-MB-231 cells after incubated on OPVBT and RGD incorporated hydrogel for 24 h. In the hydrogel, [OPVBT] = 200 μM, [RGD] = 200 μM. (d) Cell viabilities of 293T, MCF-7, and MDA-MB-231 cells in the dark or upon light irradiation after incubated on hydrogel containing different concentrations of OPVBT.

the hydrophobic and electrostatic interactions between OPVBT and cells, OPVBT could promote cell attachment and spreading in coordination with RGD molecule. Because the hydrogel also contains a MMP-cleavable sequence, after cells attachment on the hydrogel, MMP secreted from cells would locally degrade the hydrogel and OPVBT would be released and then be endocytosed by cells (Figure 4a). To validate the speculation, three cell types noted above were selected. The amounts of MMP secreted from the cells were investigated utilizing an MMP activity assay kit according to the instructions. As revealed in Figure 5b, tumor cells (MCF-7 and MDA-MB-231) have elevated MMP level as compared to normal cells (293T), which is consistent with previous reports.30,31 Afterward, imaging ability of hydrogel toward tumor cells was studied using CLSM. After different kinds of cells were added onto the hydrogel and allowed to incubate for 24 h, tumor cells (MCF-7 and MDA-MB-231) were specifically lighted up and strong OPVBT signal was observed. However, there was no obvious OPVBT fluorescent signal observed for 293T cells, confirming that the amount of MMP secreted from cells was responsible for the imaging distinction. It is noted that the cell-specific light-up imaging could be easily observed even only incubated for 12 h (Figure S3). According to the selectively light-up ability toward tumor cells, the cytotoxicity of hydrogel against different cells was

further investigated. As illustrated in Figure 5c, after incubation for 48 h in the dark, there was no obvious cytotoxicity of the hydrogel toward three types of cells. Upon light irradiation, the viability of normal cells (293T) was above 90%; however, those of tumor cells were below 40%, indicating the tumor-cell specific killing ability of the hydrogel. The results demonstrated the potential applications of hydrogel in selective image-guided PDT toward tumor cells.

4. CONCLUSIONS In summary, we have designed and synthesized a new cationic OPVBT, which could generate massive ROS upon white light irradiation. OPVBT could be endocytosed by living cells via caveolae-mediated endocytosis and further locate in mitochondria. Thus, OPVBT shows great potential as a new mitochondria-targeted photosensitizer in PDT. An MMPsensitive hydrogel system incorporated with OPVBT was constructed through Michael-type addition of cysteine-terminated oligopeptides onto 4-arm PEG-VS. The hydrophobic and electrostatic interactions between OPVBT and cells are favorable for cell attachment on the hydrogel. Due to the overexpressed MMP in tumor cells, OPVBT released from the hydrogel could selectively light up tumor cells and inhibit tumor cell growth by ROS upon light irradiation. Importantly, the MMP-sensitive hydrogel did not exhibit apparent G

DOI: 10.1021/acsbiomaterials.7b00610 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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cytotoxicity toward normal cells. The hybrid hydrogel provides a new platform for tumor-specific imaging and killing, which shows good potential for constructing targeted PDT system with reduced side effects.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00610. Inhibitors used for the study of cellular uptake mechanism; surface tensions of OPVBT at 25 °C; emission intensity of DCFH with or without OPVBT at light irradiation; CLSM images of MCF-7 cells incubated with OPVBT for different time intervals; CLSM images of 293T, MCF-7, and MDA-MB-231 cells incubated on hydrogel for 12 h (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Libing Liu: 0000-0003-4827-6009 Shu Wang: 0000-0001-8781-2535 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the National Natural Science Foundation of China (21533012, 91527306, and 21661132006), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA09030306). We thank Prof. Yilin Wang and Mr. Hua Wang for the help of surface tension measurements.

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