Degradable Semiconducting Oligomer Amphiphile for Ratiometric

Mar 15, 2017 - ... of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced ... Key Laboratory of Flexible Electronics ...
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Degradable Semiconducting Oligomer Amphiphile for Ratiometric Photoacoustic Imaging of Hypochlorite Chao Yin,†,‡,∥ Xu Zhen,‡,∥ Quli Fan,*,† Wei Huang,†,§ and Kanyi Pu*,‡ †

Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China ‡ School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457 Singapore § Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China S Supporting Information *

ABSTRACT: Upregulation of highly reactive oxygen species (ROS) such as hypochlorite (ClO−) is associated with many pathological conditions including cardiovascular diseases, neuron degeneration, lung injury, and cancer. However, real-time imaging of ClO− is limited to the probes generally relying on fluorescence with shallow tissuepenetration depth. We here propose a self-assembly approach to develop activatable and degradable photoacoustic (PA) nanoprobes for in vivo imaging of ClO−. A near-infrared absorbing amphiphilic oligomer is synthesized to undergo degradation in the presence of a specific ROS (ClO−), which integrates a π-conjugated but ClO− oxidizable backbone with hydrophilic PEG side chains. This molecular architecture allows the oligomer to serve as a degradable nanocarrier to encapsulate the ROS-inert dye and self-assemble into structurally stable nanoparticles through both π−π stacking and hydrophobic interactions. The self-assembled nanoprobe exhibits sensitive and specific ratiometric PA signals toward ClO−, permitting ratiometric PA imaging of ClO− in the tumor of living mice. KEYWORDS: photoacoustic imaging, activatable probes, polymer nanoparticles, reactive oxygen species, sensors, self-assembly, near-infrared dyes

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porphyrin-based nanostructures that can change intermolecular interactions upon heating have been demonstrated for real-time activatable PA imaging of temperature.23 Because the signals of activatable probes can evolve with the biological and pathological events, they can provide real-time information on disease status at the molecular level with low background noise.25,26 These advantages place activatable probes at the forefront of imaging, showing their crucial role in further advancing PA imaging in life science. Semiconducting polymer nanoparticles (SPNs) constructed from optically active semiconducting polymers (SPs) have emerged as an alternative class of imaging nanoagents.27−29 Because of their high absorption coefficients and controllable dimensions, fluorescent SPNs have been utilized for cell

hotoacoustic (PA) imaging capitalizes on the PA effect to combine optical excitation with ultrasonic detection, providing the advantages of deeper tissue penetration and higher spatial resolution over conventional optical imaging.1 Many exogenous light absorbers such as near-infrared (NIR) dyes,2−5 carbon nanomaterials,6−8 two-dimensional materials,9−11 porphysomes,12−15 and metallic nanoparticles16−19 have been developed as contrast agents to permit imaging of anatomic and physiological changes in diseases; however, their signals mainly result from accumulation via enhanced permeability and retention (EPR) effect or specific recognition between targeting groups and receptors on the cell surface.20 Superior to these accumulation probes, activatable PA probes that can spontaneously change their signals in response to specific molecular targets have been less developed.21−24 For example, a furin-cleavable peptide has been modified into an activatable PA probe, which can show increased PA signals to report the furin activity in the tumor of living mice.22 Besides, © 2017 American Chemical Society

Received: February 16, 2017 Accepted: March 15, 2017 Published: March 15, 2017 4174

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Figure 1. Design and synthesis of the nanoprobe for ratiometric PA imaging of ClO−. (a) Chemical structures of the SOA and NIR775 used for the synthesis of ClO−-activatable PA nanoprobe via self-assembly process and illustration of the sensing mechanism for the nanoprobe. (b) Convergent synthetic route toward the SOA (7). Reaction conditions: (i) 1,6-dibromohexane, NaH, anhydrous DMF, room temperature; (ii) NBS/anhydrous DMF, room temperature; (iii) sodium azide/anhydrous DMF, room temperature; (iv) Pd(PPh3)2Cl2, 2,6-di-tert-butylphenol, anhydrous toluene, 100 °C; (v) CuBr, N,N,N′,N″,N″-pentamethyldiethylenetriamine, THF, room temperature. (c) MALDI-TOF mass spectrum of the SOA (7).

imaging,30−33 tumor imaging,34,35 and ultrafast hemodynamic imaging36 as well as for chemiluminescence imaging of neuroinflammation37 and hepatotoxicity.38 Recently, we have found that SPNs can convert photon energy into heat in a way that can be more efficient than many other nanomaterials including carbon nanotubes and gold nanorods,39−42 permitting superior photothermal and PA imaging applications.43−48 SPNs have also been diversified into smart activatable PA probes for in vivo ratiometric imaging of reactive oxygen species (ROS)39 and pH,49 showing their versatility for PA molecular imaging. The key challenges to further organic nanoparticles in activatable PA imaging are mainly 2-fold. In terms of chemistry, preparation of water-miscible SPNs is limited to nanoprecipitation and generally requires the assistance of amphiphilic block copolymers due to the high hydrophobicity of SPs.37,42 As a result, most existing SPNs are binary micelles that tend to undergo dissociation.50 The potential issue is the escape of amphiphilic copolymer from the nanoparticles, causing the coagulation of hydrophobic components during blood circulation.51 From the viewpoint of biosafety, although SPNs are completely organic and biologically inert and no toxicity sign has been detected in living cells and mice, it generally needs a long time for them to be cleared out from animals.52 Thus, alternative molecular designs are demanded for the development of SPN-based activatable PA probes. To address these issues, we report a self-assembly approach toward organic activatable PA probes for in vivo imaging. An NIR absorbing and degradable semiconducting oligomer amphiphile (SOA) was synthesized and applied as the nanocarrier to construct activatable PA nanoprobes for ratiometric imaging of hypochlorite (ClO−). Upregulated generation of ClO−, a highly ROS, is closely associated with

many pathological conditions including cardiovascular diseases,53 neuron degeneration,54 atherosclerosis,55 lung injury,56 arthritis,57 and cancer.58 Thus, real-time in vivo imaging of ClO− can provide mechanistic information to understand the etiology of these diseases and perform diagnosis. Despite the availability of molecular probes for ClO− sensing, they generally relied on visible fluorescence as the signal readout that has the shallow tissue-penetration depth,59−61 limiting their effectiveness to in vitro studies. In contrast to fluorescent probes, the SOA-based PA nanoprobe can be excited with NIR light to emit acoustic signals with higher tissue penetration. Additionally, the degradability of the SOA-based nanoprobe differentiates itself from existing SPNs, highlighting probabilities for preclinical imaging applications.

RESULTS AND DISCUSSION To develop activatable PA nanoprobe for ratiometric imaging of ClO−, phenothiazine, a ROS-oxidizable aromatic unit,59,62 was integrated into the hydrophobic semiconducting backbone of the SOA (Figure 1a); meanwhile, poly(ethylene glycol) (PEG) was used as the side chains for the SOA to provide water solubility. Such a molecular design led to the amphiphilic nature of SOA, allowing it to act as an optically active and oxidizable nanocarrier to spontaneously encapsulate a ROSinert NIR dye (NIR775) (Figure 1a). Strong π−π stacking between the semiconducting backbone and NIR775 exists in addition to hydrophobic interaction. Such intraparticle interactions enhance the stability of nanoparticle, reduce the probability of dissociation and thus minimize the congelation of the released hydrophobic components during blood circulation. In the presence of ClO−, degradation of the SOA can occur while NIR775 remain intact, resulting in the ratiometric PA 4175

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Figure 2. Characterization of the SOA-based nanoprobe. (a) Representative TEM of the nanoprobe. (b) DLS of the nanoprobe. (c) Average hydrodynamic diameters of the nanoprobe stored in PBS for different time periods. (d) UV−vis absorption spectra of the nanoprobe (30 μg mL−1) upon addition of ClO− at intervals of 1 μM. Inset: photograph of the nanoprobe solution before and after addition of ClO− (11 μM). (e) Absorption ratiometric signals of the nanoprobe (Ab780/Ab680) as a function of ClO− concentration. The red line represents linear fitting from [ClO−] = 0 to 10 μM. (f) Absorption ratiometric responses (Ab780/Ab680) of the nanoprobe (30 μg mL−1) toward different ROS (11 μM) in PBS buffer. (g) Average diameter of the nanoprobe incubated with ClO− at different concentrations. The arrow indicates the average diameter of the NIR775 nanoparticles prepared by nanoprecipitation without SOA (the bare NIR775 nanoparticles). (h) Fluorescence spectra of the nanoprobe treated with ClO− from 0 (1) to 11 μM (2) and the bare NIR775 nanoparticles (3) (λex = 720 nm). (i) Fluorescence images of the nanoprobe without (1) and with (2) ClO− treatment as well as the bare NIR775 nanoparticles (3). Images were obtained from IVIS with excitation at 710 nm and emission at 780 nm. The concentration of the nanoprobe was fixed at 30 μg mL−1. The error bars represent standard deviations (SD) of three separate measurements.

and 3.86 ppm, respectively, indicating the successful linkage between the two segments. The resonance peaks of the MPEG protons were located at 3.82−3.37 ppm, and the resonance peaks of alkyl chain proton were at 1.78−1.61, 1.38−1.21, and 0.92−0.79 ppm (Figure S1, Supporting Information). The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum (Figure 1c) revealed that the molecular weight of the maximum peak was determined to be 11178.12 Da, nearly identical to the theoretical value of SOA (7). Moreover, a family of peaks were clearly detected in the spectrum, and the interval between two adjacent peaks was 44.21 Da, corresponding to the mass of the oxyethylene repeat unit of PEG. These data clearly proved the successful synthesis and correct structure of the SOA (7). Self-assembly of the SOA in water in the presence of NIR775 led to the ClO−-activatable nanoprobe (Figure 1a). Transmission electron microscopy (TEM) indicated the spherical morphology of the nanoprobe (Figure 2a). Dynamic light scattering (DLS) showed the average hydrodynamic size of the nanoprobe at 33.8 ± 1.2 nm with a narrow polydispersity index (PDI) of 0.39 (Figure 2b), which remained almost the same even after storage in phosphate-buffered saline (PBS, pH = 7.4)

signals. The good water-solubility and PEG-passivated surface of the nanoprobe also permit ratiometric PA imaging of ClO− in tumors of living mice after systemic administration of the nanoprobe. The SOA (7) was synthesized via a convergent route (Figure 1b). Phenothiazine (1) was first alkylated with 1,6-dibromohexane in the presence of sodium hydride (NaH) to obtain compound 2. Bromination of 2 was conducted carefully using N-bromosuccinimide (NBS) to yield a single bromine substituted compound 3. The alkyl bromide in compound 3 was substituted with azide through nucleophilic substitution to produce compound 4. Stille coupling reaction was then conducted between compounds 5 and 4, yielding the azide substituted semiconducting oligomer 6. At last, click chemistry was used to conjugate the methoxy-end-capped PEG (MPEG)alkyne (Mn 5000) to the side chain of 6, affording the SOA (7). The correct structures of all the intermediates and the final product were confirmed by 1H NMR and mass spectra. For the SOA, the resonance signal assigned to endo thienyl protons of diketopyrrolopyrrole (DPP) shifted noticeably to 8.90 ppm, and the signals of the benzene ring and methylene-H attached to the nitrogen of phenothiazine were detected at 7.54−6.82 4176

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Figure 3. In vitro PA imaging of ClO−. (a) Representative PA spectra ranging from 680 to 860 nm of the nanoprobe (30 μg mL−1) in the absence and presence of ClO− (11 μM). (b) Quantification of the ratiometric PA signals (PA780/PA680) of the nanoprobe (30 μg mL−1) as a function of ClO− concentration. The red line represents linear fitting from [ClO−] = 0 to 10 μM. (c) Ratiometric PA responses (PA780/PA680) of the nanoprobe (30 μg mL−1) toward different ROS (11 μM) in PBS buffer. (d) PA images of the nanoprobe upon ClO− treatment at different concentrations. The samples were placed in the plastic tubes to conduct the PA imaging tests at 680 and 780 nm, which are indicated in pseudo green and red, respectively. e) PA images of the nanoprobe upon treatment of H2O2, •OH, 1O2, and ONOO− at the same concentration (11 μM). A pulsed laser was turned to 680 or 780 nm for ratiometric imaging. The error bars represent standard deviations of three separate measurements.

tration of ClO− was observed with the limit of detection of 0.70 μM. At the saturation point, Ab780/Ab680 (6.22 ± 0.24) for ClO− was 4.06-fold higher than that at the initial state and other ROS (Figure 2f). The kinetics of ClO−-induced optical change of the nanoprobe was investigated, showing a nearly complete probe activation within 150 s (Figure S4, Supporting Information). In view of the high level of ClO− under inflammatory conditions (20−400 μM per hour by activated neutrophils)63 and the inflammatory microenvironment of tumor,64 the nanoprobe should be able to detect aberrant level of ClO− in living mice. The sensing mechanism was studied by conducting the model reaction between compound 6 and ClO− (Figure S5, Supporting Information). After the reaction, mass spectroscopy was able to detect 2-(10-(6-azidohexyl)-5,5-dioxido-10Hphenothiazin-3-yl)-2-oxoacetic acid (11) and 2,2′-(2,5-bis(2ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole1,4-diyl)bis(2-oxoacetic acid) (12) as the final reaction products. According to the reported literatures,59,65,66 the sulfur atoms in thiophene and phenothiazine are sensitive to oxidation, particularly to ClO−. First, the sulfur atoms are oxidized by ClO− to form the corresponding sulfones. Then, further oxidation of thiopene 1,1-dioxide causes the extrusion of SO2 and the attachment of hydroxyl groups, leading to the intermediate 9. Nine can undergo further oxidation to produce compound 10. At last, 10 is cleaved, yielding 11 and 12 as the final products. Such a ClO−-induced degradation is not sensitive to pH as shown by the pH-independent response of the nanoprobe toward ClO − (Figure S6, Supporting Information). This proved the ability of the nanoprobe to detect ClO− regardless of pH at disease sites. The structural evolution of the nanoprobe upon treatment of ClO− was further investigated by monitoring the size and fluorescence of the nanoprobe. DLS exhibited a continuous change in the average diameter of the nanoprobe, increasing

for 30 d (Figure 2c). This indicated the high structural stability of the nanoprobe, probably due to the good water solubility provided by PEG chains as well as the structural integrity provide by strong π−π stacking and hydrophobic interactions between the semiconducting part of the SOA and NIR775. The optical properties of the SOA and the nanoprobe were investigated. Without NIR775, the SOA itself had a maximum absorption peak at 596 nm with a shoulder at 650 nm in PBS (Figure S2a, Supporting Information). The maximum emission peak of the SOA was at 810 nm in PBS (Figure S2b, Supporting Information), showing a very low fluorescence intensity with the quantum yield of ∼0.1%. With the incorporation of NIR775, the nanoprobe had a new band at 784 nm (Figure S2a, Supporting Information), which can be attributed to the absorption of NIR775 according to the absorption band of PEG-b-PPG-b-PEG-encapsulated NIR775 nanoparticles in PBS solution (Figure S2a, Supporting Information). In order to obtain the ideal ratiometric PA response, the optimal doping amount of NIR775 was fixed at 0.4w/w%, where the absorption intensities at 780 and 680 nm were nearly identical. Changes in the absorption spectra of the nanoprobe upon addition of different ROS were then studied in pH = 7.4 PBS buffer. The absorption peak at 596 nm assigned to the SOA within the nanoprobe decreased gradually with increased concentration of ClO−, while the absorption peak assigned to NIR775 at 784 nm remained almost the same (Figure 2d). In contrast, the absorption profile showed no change for other ROS including H2O2, •OH, 1O2, and ONOO− (Figure S3, Supporting Information), indicating the high selectivity of the nanoprobe to ClO−. The spectral change of the nanoprobe toward ClO− allowed us to quantify the signals using the ratio of the absorption intensity at 780 to that at 680 nm (Ab780/ Ab680) (Figure 2e). A good linear correlation between the ratiometric absorption signal (Ab780/Ab680) and the concen4177

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Figure 4. In vivo PA imaging of ClO−. (a) Representative PA image of a subcutaneous 4T1 tumor in a nude mouse before and 2, 4, 6, 8, and 24 h after intravenous administration of the nanoprobe (50 μg per mouse). The representative PA maximum intensity projection (MIP) images with axial view are demonstrated. (b) Quantification of the PA intensity increment at 680 (ΔPA680) and 780 nm (ΔPA780) as a function of time postinjection of the nanoprobe. (c) Ratiometric PA signals (ΔPA780/ΔPA680) as a function of postinjection time. The pulsed laser was tuned to 680 or 780 nm for PA mapping. The error bars represent standard deviations of three separate measurements (n = 3).

increased linearly with the concentration of ClO− (Figure 3b), confirming the probability for quantification of ClO−. The limit of detection was determined to be 1.3 μM. At the saturation point, PA780/PA680 (7.2 ± 0.18) was 8.3-fold higher than that for its initial unactivated state (0.87 ± 0.3) as well as for other ROS (Figure 3c). In addition to such a selective PA response toward ClO−, the nanoprobe was proven to have high photostability (Figure S8, Supporting Information) and low cytotoxicity (Figure S9, Supporting Information). These data thus reflected the potential of the nanoprobe for in vivo PA imaging of ClO−. The capability of the nanoprobe for in vivo imaging of ClO− was validated using the subcutaneous 4T1 xenograft tumor model. After systemic administration of the nanoprobe, the PA images were recorded at 680 and 780 nm, which were indicated in pseudo green and red colors, respectively (Figure 4a). As tumors had the intrinsic weak PA signals due to the absorption of oxy- and deoxyhemoglobins, the PA intensity increment (ΔPA, defined as the PA intensity after injection of the nanoprobe deducted by the tissue intensity before injection of the nanoprobe) was used to evaluate the ratiometric PA signals (ΔPA780/ΔPA680) so as to minimize the tissue interference. ΔPA780 increased significantly over time after injection of the nanoprobe and reached the maximum at 6 h postinjection, while ΔPA680 increased slightly (Figure 4b). As the signal at 780 nm is insensitive to ROS, the strong signal increment at 780 nm should be assigned to the EPR effect of the nanoprobe owing to its small size. This was also confirmed by ex vivo biodistribution data (Figure S10, Supporting Information), showing the slightly lower signal from tumors as compared with that from livers. The weak signal increment at 680 nm implied that the degradation of the nanoprobe by ClO− in the tumor environment occurred simultaneously with the probe accumulation. As a result of such signal evolution at two channels, the ratiometric PA signals (ΔPA780/ΔPA680) gradually increased over time and reached the maximum at 6 h postinjection

from 33.8 ± 1.2 nm at its initial unactivated state to 67.8 ± 1.4 nm after activation by ClO− (Figure 2g). This size was close to that of the NIR nanoparticles prepared by nanoprecipitation without SOA (the bare NIR775 nanoparticles) (71.3 ± 2.3 nm). Furthermore, the fluorescence of the nanoprobe at 786 nm assigned to NIR775 gradually decreased upon addition of ClO− from 0 to 11 μM (Figure 2h). When the fluorescence change saturated, the fluorescence intensity at 786 nm was identical to the bare NIR775 nanoparticles at the same optical concentration (Figures 2h,i). These phenomena implied that ClO−-induced degradation facilitated the aggregation of hydrophobic NIR775 because the amphiphilic nanocarrier (SOA) no longer existed. At the final stage, the aggregation of NIR775 reached the status that was similar to the bare NIR775 nanoparticles. The ability of the nanoprobe for ratiometric PA imaging of ClO− was first examined in solution. The nanoprobe emitted PA signals ranging from 680 to 860 nm with two peaks at 680 and 780 nm, assigned to SOA and NIR775, respectively. Moreover, the PA intensity of the nanoprobe at 680 nm was nearly identical to that of the SOA itself (Figure S7b, Supporting Information). Upon addition of ClO−, the PA intensities of the nanoprobe at 680 nm gradually decreased, but the peak at 780 nm remained nearly unchanged (Figure 3a). The PA images of the nanoprobe solutions at different amounts of ClO− were recorded at two wavelengths (680 or 780 nm), and the images were indicated in pseudo green and red for 680 and 780 nm, respectively. When the concentration of ClO− was increased, the red signals remained nearly unchanged; however, the green signals exhibited continuous attenuation and eventually vanished (Figure 3d). In contrast, in the presence of other ROS including H2O2, •OH, 1O2, and ONOO−, both red and green signals remained unchanged (Figure 3e). This dual-wavelength spectral response allowed us to conduct ratiometric PA imaging of ClO−. Similar to the ratiometric absorption, the ratiometric PA signal (PA780/PA680) also 4178

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ACS Nano (Figure 4c). At this time point, ΔPA780/ΔPA680 was ∼1.47-fold (plateau, 3.15 ± 0.12) higher than that at 2 h postinjection (2.15 ± 0.1). This data clearly proved the activation of the nanoprobe by ClO− in the tumor microenvironment of living mice.

Synthesis of 10-(6-Bromohexyl)-10H-phenothiazine (2). A 250 mL round-bottom flask with magnetic stirring bar was charged with a mixture of sodium hydride (60% in mineral oil, 1.35 g, 33.75 mmol) and 1,6-dibromohexane (18.3 g, 75 mmol) under an argon atmosphere. Then 5 g of 10H-phenothiazine (dissolved in 25 mL of anhydrous DMF) was slowly added into the round-bottom flask. The mixture was stirred at room temperature overnight, which was poured into water and extracted with diethyl ether. The separated organic layer was dried over Na2SO4 and the solvent removed using rotary evaporation. The crude product was purified by column chromatography (silica gel, petroleum ether) to afford 7.75 g (85%) of 2 as a colorless oil. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 7.25−7.10 (m, 4H), 7.05−6.88 (m, 4H), 3.86 (t, 2H), 3.48 (t, 2H), 1.81−1.62 (m, 4H), 1.38 (s, 4H). ESI-MS m/z: 364.05 [M + H]+. Synthesis of 3-Bromo-10-(6-bromohexyl)-10H-phenothiazine (3). N-Bromosuccinimide (2.22 g, 12.48 mmol) was slowly added to a solution of 2 (4.53 g, 12.48 mmol) in anhydrous DMF (25 mL). The mixture was stirred at room temperature overnight, which was poured into water and extracted with diethyl ether. The organic layer was washed with brine and then dried over Na2SO4. After the solvent was removed under reduced pressure, the pure product was obtained as a light-yellow oil (4.12 g, 74.9%) by column chromatography (silica gel, petroleum ether/dichloromethane = 20:1). 1H NMR (300 MHz, DMSO-d6, ppm) δ: 7.37−7.30 (m, 2H), 7.24−7.11 (m, 2H), 7.04− 6.89 (m, 3H), 3.82 (t, 2H), 3.46 (t, 2H), 1.79−1.57 (m, 4H), 1.41− 1.31 (m, 4H). ESI-MS m/z: 442.21 [M + H]+. Synthesis of 10-(6-Azidohexyl)-3-bromo-10H-phenothiazine (4). Sodium azide (1.82 g, 28 mmol) was added to a solution of 3 (4.12 g, 9.34 mmol) in anhydrous DMF (25 mL), and the mixture was stirred at room temperature for 24 h. After the solvent was removed by rotary evaporation, the mixture was extracted with water and dichloromethane. The pure product can be obtained without further purification after removal of the solvent for the organic layer (3.6 g, 95.36%). 1H NMR (300 MHz, DMSO-d6, ppm) δ: 7.39−7.31 (m, 2H), 7.24−7.12 (m, 2H), 7.05−6.89 (m, 3H), 3.84 (t, 2H), 3.26 (t, 2H), 1.71−1.60 (m, 2H), 1.53−1.43 (m, 2H), 1.40−1.29 (m, 4H). ESI-MS m/z: 405.12 [M + H]+. Synthesis of 3,6-Bis(5-(10-(6-azidohexyl)-10H-phenothiazin-3-yl)thiophene-2-yl)-2,5-bis(2-ethylhexyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (6). Compound 4 (3 g, 7.4 mmol), 2,5-bis(2ethylhexyl)-3,6-bis(5-(trimethylstannyl)thiophene-2-yl)-2,5dihydropyrrolo[3,4-c]pyrrole-1,4-dione (2.87 g, 3.38 mmol), Pd(PPh3)2Cl2 (0.24 g, 0.34 mmol), and 2,6-di-tert-butylphenol (35.9 mg, 0.174 mmol) were placed in a 50 mL round-bottom flask, and the reaction vessel was degassed and purged with N2. Then 20 mL of anhydrous toluene was injected into the mixture, which was subjected to three freeze−pump−thaw cycles to remove O2. The mixture was vigorously stirred at 100 °C for 24 h. After the organic solvent was distilled, the crude product was purified by column chromatography (silica gel, petroleum ether/dichloromethane = 1:1.5) to give a blue solid (1.78 g, 45.1%). 1H NMR (300 MHz, CDCl3, ppm) δ: 8.93 (s, 2H), 7.62−7.31 (m, 7H), 7.20−7.11 (m, 3H), 7.00−6.68 (m, 6H), 4.07 (d, 4H), 3.89 (t, 4H), 3.24 (t, 4H), 1.97−1.89 (m, 2H), 1.88− 1.76 (m, 4H), 1.38−1.20 (m, 28H), 0.94−0.76 (m, 12H). MALDITOF, m/z: calcd 1168.50, found 1168.31 (M+). Synthesis of SOA (7). The final product SOA (7) was synthesized through a “click” reaction. Compound 6 (20 mg, 0.017 mmol), MPEG-alkyne (Mw 5000, 213.7 mg, 0.043 mmol), CuBr (12 mg, 0.0833 mmol), and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 110 μL) were mixed in a 25 mL round-bottom flask, which was degassed and purged with N2. Then 15 mL of THF was injected to the flask, and the mixture was stirred at room temperature for 2 days. After the solvent was removed, an appropriate amount of deionized water was added to disperse the mixture under ultrasonic conditions. Thereafter, the resulting solution was purified by dialysis against water for 2 days with a dialysis bag (Mw 50000 Da) to remove the small molecules. Finally, the solution was extruded using a syringe through 0.22 μm membranes and dried by freeze-drying to obtain blue floccules (152.6 mg, 79.9%). 1H NMR (300 MHz, CDCl3, ppm) δ: 8.90 (s, 2H), 7.54−7.30 (m, 6H), 7.18−6.82 (m, 10H), 4.05 (m, 4H),

CONCLUSIONS In conclusion, we have demonstrated a self-assembly approach based on organic semiconducting amphiphile toward activatable and degradable PA nanoprobes for in vivo imaging. An NIR-absorbing amphiphilic SOA was designed and synthesized to undergo degradation in the presence of a specific ROS (ClO−) by integrating a π-conjugated but ClO−-oxidizable backbone with hydrophilic PEG side chains. Such a molecular architecture allowed the SOA to serve as a degradable nanocarrier to encapsulate the hydrophobic ROS-inert NIR775 and self-assemble into structurally stable nanoparticles in biologically relevant media. The SOA-based nanoprobe exhibited sensitive and specific ratiometric PA signals toward ClO−, showing ∼8.3-fold enhancement in the ratiometric PA signals upon activation. With good biocompatibility, small size, and high structural stability, the nanoprobe allowed for ratiometric PA imaging of ClO− in the tumor of living mice. As the nanoprobe had an ideal biodistribution, it should be promising for imaging of ClO− in other pathological conditions such as chronic inflammation, cardiac ischemia, and neurodegenerative diseases. Our study thus provides a generation of organic degradable PA nanoagents with flexibility for advanced preclinical molecular imaging. The degradable molecular design can be generalized for PA imaging of other targets of interest such as oxidative enzymes if enzyme-degradable components can be utilized as the building blocks for the construction of optically active macromolecule. In view of the self-assembly feature, the design concept should be applicable to encapsulate other components such as inorganic nanoparticles, drug molecules, and sensing agents, permitting other applications such as multimodality imaging and theranostics. EXPERIMENTAL SECTION Chemicals. All chemicals were obtained from Sigma-Aldrich unless otherwise stated. Sodium hypochlorite (NaOCl, 5% aqueous solution) and potassium superoxide (KO2) were purchased from Aladdin (Shanghai, China). Milli-Q water was supplied by Milli-Q Plus System (Millipore Corp., Breford, MA). Materials Characterization. NMR spectra were recorded on a Bruker Ultra Shield Plus 300 MHz NMR. LC−MS was performed on Thermo LCQ Fleet LC-MS with ESI mode (America). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF-MASS) was performed on a Bruker autoflex under the reflector mode for data acquisition. TEM images were obtained on a JEM 1230 transmission electron microscope with an accelerating voltage of 200 kV. DLS was performed on the Malvern ZetaSizer Nano S. UV−vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer. PA Imaging Instrumentation. A commercial Endra Nexus128 PA tomography system (Endra Inc., Ann Arbor, MI) was used for PA imaging study. The system houses a tunable nanosecond pulsed laser (7 ns pulses, 20 Hz pulse repetition frequency, 7 mJ/pulse on the animal surface, wavelength range (680−950 nm), 128 unfocused ultrasound transducers with 5 MHz center frequency and 3 mm diameter) arranged in a hemispherical bowl filled with water, animal tray on top of the bowl, data acquisition/reconstruction console, servo motors for 3D rotation of the bowl, and a temperature monitor of the water bath. 4179

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ACS Nano 3.86 (m, 4H), 3.82−3.38 (m, 875H), 3.37 (s, 6H), 1.78−1.61, 1.38− 1.21, 0.92−0.79 (chemical shifts of the proton in alkyl chains). MALDI-TOF MS (the maximum peak, m/z): 11178.12 (M+). Preparation of Nanoprobe. SOA (5.0 mg) and NIR775 (0.02 mg) were dissolved in 1.0 mL of THF and then swiftly dropped into water (10 mL) under sonication. THF was then removed by nitrogen blowing on the solution surface under stirring at room temperature. After filtration through a 0.22 μm filter, a bright blue aqueous solution was obtained. Then the resultant solution was freeze-dried and reconstituted in PBS for further experiments. Cell Viability. In vitro cytotoxicity of the nanoprobe was determined in Hela cells by the MTT assay. Hela cells were incubated on 96-well plate in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2 humidified atmosphere for 24 h, and 0.5 × 104 cells were seeded per well. Cells were then cultured in the medium supplemented with the indicated doses of the nanoprobe for 24 h. The final concentrations of the nanoprobe in the culture medium were fixed at 5, 10, 20, 30, 40, and 50 μg mL−1 in the experiment. Addition of 10 μL of MTT (0.5 mg mL−1) solution to each well and incubation for 3 h at 37 °C was followed to produce formazan crystals. Then the supernatant was removed, and the products were lysed with 200 μL of DMSO. The absorbance value was recorded at 590 nm using a microplate reader. The absorbance of the cells treated with the same volume of PBS was used as a control, and its absorbance was used as the reference value for calculating 100% cellular viability. PA Spectroscopic Measurements. For PA spectroscopic measurements, an optical parametric oscillator, OPO (Continuum, Surelite), pumped by a Q-switched 532 nm Nd:YAG laser was used as an excitation source. The solution of the nanoprobe treated by ClO− with different concentrations was place inside a low-density polyethylene (LDPE) tube with an inner diameter (i.d.) of 0.59 mm and outer diameter (o.d.) of 0.78 mm. The sample containing an LDPE tube and the single-element ultrasound transducer, UST (V323-SU/ 2.25 MHz, 13 mm active area, and 70% nominal bandwidth, Panametrics), were immersed in water medium for coupling of photoacoustic signals to UST. The LDPE tube was irradiated with wavelengths ranging from 680 to 970 nm with 10 nm increments. Respective PA signals were collected using the UST, and these signals were subsequently amplified with a gain of 50 dB and band-pass filtered (1−10 MHz) by a pulser/receiver unit (Olympus-NDT, 5072PR). Finally, the output from the pulser/receiver unit was digitized with a data acquisition card (GaGe, compuscope 4227) operated at 25 MHz, and the acquired signals were stored in the computer. Peak-to-peak voltage of the PA signal was then normalized with the laser energy for each wavelength and was plotted against the wavelength to generate the PA spectrum. For the nanoprobe in the absence and presence of ClO− (11 μM), the full spectra from 680 to 860 nm were recorded. For the nanoprobe treated by ClO− with other different concentrations (2, 3, 4, 5, 6, 7, 8, 9, and 10 μM), the PA intensities at 680 and 780 nm were detected, respectively. Tumor Mouse Model. All animal experiments were performed in compliance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC), Sing Health. To establish tumors in 6-week-old female nu/nu mice, 2 million 4T1 cells suspended in 50 mL of 50 v/v% mixture of matrigel in supplemented Dulbecco Modified Eagle Medium (DMEM, 10% fetal bovine serum, 1% pen/ strep, 100 U/mL penicillin, and 100 μg mL−1 streptomycin) were injected subcutaneously in the shoulders of the mouse. Tumors were grown until a single aspect was ∼7 mm (approximately 10−15 days) before used for PA imaging. In Vivo PA Imaging of ClO−. 4T1 tumor xenografted nude mice were anesthetized using 2% isoflurane in oxygen, and a catheter was applied to the tail vein. They were placed in the Endra Nexus128 PA imaging system and were scanned to determine the endogenous signal of tumors at 680 and 780 nm before systemic administration with the nanoprobe (200 μL, 0.25 mg mL−1) (n = 3) and after different postinjection time periods. Mice treated with saline (200 μL) (n = 3) were set as the control. Data was acquired at 680 and 780 nm through a continuous model that took 12 s to obtain one data set. The three-

dimensional PA image was reconstructed off-line using data acquired from all 128 transducers at each view and a back-projection algorithm. The algorithm corrects for pulse-to-pulse variations in the laser intensity and small changes in the temperature that affect acoustic velocity in the water. Data Analysis. PA signal intensities were measured by region of interest (ROI) analysis using OsiriX. Results are expressed as the mean ± SD deviation unless otherwise stated. All statistical calculations were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA).

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01092. 1 H NMR spectrum of 7, photophysical properties of SOA and the nanoprobe, absorption responses of the nanoprobe toward different ROS, kinetics of the hypochlorite-induced optical change of the nanoprobe, proposed mechanism of the ClO−-induced degradation of 7, and effect of pH on the ratiometric absorption of the nanoprobe as well as the absorption stability, cytotoxicity studies, and biodistribution of the nanoprobe (Figures S1−S10) (PDF)

AUTHOR INFORMATION Corresponding Authors

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

Quli Fan: 0000-0002-9387-0165 Wei Huang: 0000-0001-7004-6408 Kanyi Pu: 0000-0002-8064-6009 Author Contributions ∥

C.Y. and X.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS K.P. thanks Nanyang Technological University (Start-Up grant: NTU-SUG: M4081627.120) and the Singapore Ministry of Education (Academic Research Fund Tier 1: RG133/15 M4011559 and Academic Research Fund Tier 2 MOE2016T2-1-098) for financial support. W.H. and Q.F. thank the National Basic Research Program of China (No. 2012CB933301), the National Natural Science Foundation of China (Nos. 21674048, 21574064, 61378081, 11404219, and 61505076), the Synergetic Innovation Center for Organic Electronics and Information Displays, and the Natural Science Foundation of Jiangsu Province of China (Nos. BZ2010043, NY211003, and BM2012010) for financial support. REFERENCES (1) Wang, L. V.; Hu, S. Photoacoustic Tomography: In vivo Imaging from Organelles to Organs. Science 2012, 335, 1458−1462. (2) Zhang, Y.; Cai, X.; Wang, Y.; Zhang, C.; Li, L.; Choi, S. W.; Wang, L. H. V.; Xia, Y. A. Noninvasive Photoacoustic Microscopy of Living Cells in Two and Three Dimensions Through Enhancement by a Metabolite Dye. Angew. Chem., Int. Ed. 2011, 50, 7359−7363. (3) Fan, Q.; Cheng, K.; Yang, Z.; Zhang, R.; Yang, M.; Hu, X.; Ma, X.; Bu, L.; Lu, X.; Xiong, X.; Huang, W.; Zhao, H.; Cheng, Z. Perylene-Diimide-Based Nanoparticles as Highly Efficient Photo4180

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