Amphiphilic Semiconducting Oligomer for Near-Infrared Photoacoustic

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Amphiphilic Semiconducting Oligomer for NearInfrared Photoacoustic and Fluorescence Imaging Chao Yin, Xu Zhen, Hui Zhao, Yufu Tang, Yu Ji, Yan Lyu, Quli Fan, Wei Huang, and Kanyi Pu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02014 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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ACS Applied Materials & Interfaces

Amphiphilic Semiconducting Oligomer for Near-Infrared Photoacoustic and Fluorescence Imaging Chao Yin,† Xu Zhen,‡ Hui Zhao,† Yufu Tang,† Yu Ji,† Yan Lyu, ‡ 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, Singapore, 637457

§

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

*

Address correspondence to: [email protected]; [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Semiconducting polymer nanoparticles (SPNs) have emerged as an alternative class of optical nanoagents for imaging applications. However, the general preparation method for SPNs is nanoprecipitation, which is likely to encounter the issue of nanoparticle dissociation. We herein report non-dissociable near-infrared (NIR)-absorbing organic semiconducting nanoparticles for in vivo photoacoustic (PA) and fluorescence imaging. The nanoparticles are self-assembled from an amphiphilic semiconducting oligomer (ASO) that has a hydrophobic semiconducting oligomer backbone attached by hydrophilic poly(ethylene glycol) (PEG) side chains. ASO has higher structural stability and brighter PA signals as compared with its counterpart nanoparticles (SON) synthesized by nanoprecipitation. The small size and PEG-passivated surface of ASO allow it to passively target to and efficiently accumulate in the tumor of living mice, permitting tumor imaging with high signal-to-background ratios. Our study provides new NIR-absorbing organic nanoparticles for PA and fluorescence imaging, which also have the potential to be used as drug carrier for theranostics. KEYWORDS: semiconducting oligomer, self-assembly, nanoparticles, photoacoustic imaging, fluorescence imaging

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INTRODUCTION Photoacoustic (PA) imaging is a noninvasive and nonionizing imaging technique that combines optical excitation with ultrasonic detection for deeper imaging depth and higher spatial resolution as compared with conventional optical imaging.1 Because not all the diseases express endogenous light absorbers such as hemoglobin and melanin,2 exogenous imaging agents with near-infrared (NIR) extinction are often required for PA imaging. Till now, metallic nanoparticles,3-5 carbon nanotubes,6,7 NIR dyes,8,9

fluorescence

proteins,10,11

two-dimensional

materials,12,13

and

porphysomes14,15 have been widely developed into the contrast agents for PA imaging. Although all these materials have their own merits, they have certain drawbacks, for instance, difficulty in biodegradation and long-term toxicity for metallic nanoparticles, generally poor photostability for NIR dyes and fluorescence proteins, broad PA spectral profiles for carbon nanotubes and two-dimensional materials, and phototoxicity for porphysomes. Thus, alternative materials capable of converting photon energy into acoustic signals are highly desired for further advancing PA imaging in life science. Semiconducting polymer nanoparticles (SPNs) made from semiconducting polymers (SP) have emerged as a new category of optical imaging nanomaterials.16-19 In view of their unique advantages such as good biocompatibility, excellent optical properties and structural versatility, fluorescent SPNs have been widely employed for cell tracking,20,21 tumor imaging22,23 and ultrafast hemodynamic imaging24 as well as for chemiluminescence imaging of hepatotoxicity25 and neuroinflammation.26 3



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Furthermore, SPNs have been revealed to efficiently convert photon energy into heat, allowing for photothermal therapy27,28 and real-time activation of neurons.29 The high photothermal conversion efficiencies of SPNs make them good agents for PA imaging, which have been validated for in vivo imaging of lymph nodes,30 tumor,31-37 reactive oxygen species (ROS)30 and pH.38 Despite the advantages of SPNs for PA imaging, the main preparation method of SPNs is limited to nanoprecipitation: incorporate semiconducting polymers into amphiphilic block copolymers such as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-b-PPG-b-PEG),26 poly(styrene-co-maleic anhydride) (PSMA)22 and polystyrene graft ethylene oxide functionalized with carboxylic acid (PS-PEG-COOH).39 Therefore, most existing SPNs have the potential issues of slow dissociation as they are binary micelles.40 Such dissociation could result in aggregation during blood circulation, resulting in changed optical properties and poor biodistribution.41,42 Thus, other designs to apply SPNs for PA imaging are desired. In this study, we report the design and synthesis of an amphiphilic semiconducting oligomer (ASO) for PA and fluorescence dual-modal imaging. ASO is composed of a hydrophobic semiconducting backbone attached by hydrophilic poly(ethylene glycol) (PEG) side chains. The semiconducting backbone serves as the signal source that efficiently converts photon energy into fluorescence and PA signals, while PEG side chains provide water-solubility and shield the hydrophobic backbone to reduced non-specific interactions with plasma proteins. Such a functional amphiphilic nature 4


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endows ASO with the ability to self-assemble into water-soluble nanoparticles in aqueous solution, and thus overcome the dissociation issue of SPNs. In the following, the design rationale and synthesis of ASO are described, followed by comparison studies of its optical properties and stability with the corresponding nanoparticles prepared from nanoprecipitation. Then, the PA properties of ASO are discussed. At last, the proof-of-concept PA and fluorescence imaging application is demonstrated using the xenograft tumor mouse model.

RESULTS AND DISCUSSION ASO was synthesized via Pd-catalyzed Suzuki coupling reaction followed by copper(I)-catalyzed

“click”

reaction

(Scheme

1a).

3,6-Bis(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole -1,4-dione (TPADPP) was chosen as the semiconducting backbone because DPP-based materials generally have excellent optical and photothermal properities.31 First,

Suzuki

reaction

N,N-Diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline

between (1)

and

2,5-Bis(6-bromohexyl)-3,6-bis(5-bromothiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrr ole-1,4-dione (2) in the presence of Pd(0) and K2CO3 yielded the semiconducting oligomer 3. Then, the alkyl bromides of 3 were converted into azide through nucleophilic substitution using NaN3, producing the semiconducting oligomer 4. Finally, “click” chemistry was employed to conjugate alkyne-end-capped PEG (average M.W. 5000) as the side chains to 4, affording the final product ASO (5). 5



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The correct structures of all the intermediates and ASO were confirmed via 1H NMR and mass spectra. In the 1H NMR spectrum of ASO (Figure S5, Supporting Information), the resonance signal attributed to endo thienyl protons of DPP was found at 8.89 ppm, and the signals of benzene ring within triphenylamine was found at 7.61-6.82 ppm. This indicated the successful linkage between DPP and triphenylamine moieties. In addition, the resonance peaks of the PEG protons were located at 3.82-3.37 ppm, and the resonance peaks of alkyl chain proton were at 1.50-0.75

ppm.

Matrix

assisted

laser

desorption/ionization

time-of-flight

(MALDI-TOF) mass spectrum showed the maximum peak of the molecular weight at 11029.39 Da (Scheme 1b), nearly identical to the theoretical value of ASO (11036.40 Da). Furthermore, a series of peaks with the interval of ~44.0 Da for two adjacent peaks were identified, which was identical to the mass of the ethylene oxide repeat unit. These results clearly supported the successful synthesis of ASO (5).

Scheme 1. (a) Design and synthesis of the amphiphilic semiconducting oligomer (ASO, 5). Reaction conditions: (i) Pd(PPh3)4, K2CO3, toluene, 100 oC. (ii) Sodium azide, anhydrous DMF/THF, room temperature. (iii) CuBr, N, N, N', N'',

6


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N''-pentamethyldiethylenetriamine, THF, room temperature. (b) Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum of ASO.

ASO could be directly dissolved in aqueous solution and spontaneously self-assemble into nanoparticles (Figure 1b). The corresponding semiconducting oligomer nanoparticle (SON) was prepared as the control via a nanoprecipitation method

using

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-5000] (DSPE-mPEG5000) and the semiconducting oligomer 4 (Figure 1a). Both ASO and SON showed the similar sapphire color in phosphate buffered saline (PBS, pH = 7.4) (Figure 1c) Transmission electron microscopy (TEM) indicated the spherical morphology for both ASO and SON (Figure 1d). Dynamic light scattering (DLS) showed the average hydrodynamic size of ASO was 23.84 ± 1.9 nm, smaller than SON (48.41 ± 2.4 nm) (Figure 1e). No precipitation and obvious changes in the average diameter were observed for ASO after storage in PBS (pH = 7.4) for 30 days (Figure 1f), indicating its high stability in aqueous solution. In contrast, dissociation was observed for SON, which was witnessed by a new peak in DLS spectrum ranging from 147 to 159 nm after storage in PBS (pH = 7.4) for 30 days (Figure 1g). These data indicated superior stability of ASO over SON in aqueous solution. This was mainly due to the amphiphilic structure of ASO that comprised the semiconducting chromophores covalently linked with PEG segments; in contrast, SON had the hydrophobic semiconducting chromophores non-covalently encapsulated by the

7



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amphiphilic polymer, which had the issue of dissociation and leakage of the hydrophobic component.

Figure 1. (a) Scheme illustration of preparation of SON via nanoprecipitation using DSPE-mPEG5000 and compound 4. (b) The illustration of the synthesis for ASO nanoparticles via self-assembly process of 5 in aqueous solution. (c) Photograph of the nanoparticle solutions. From left to right: SON, ASO. (d) Representative transmission electron microscopy (TEM) of ASO (left) and SON (right). (e) Dynamic light scattering (DLS) of ASO and SON. (f) Average hydrodynamic diameters of the ASO stored in PBS for different time periods (0 to 30 days). (g) Representative DLS of SON prepared immediately and 30 days post-prepared in PBS (pH = 7.4). The optical properties of ASO were investigated and compared with SON by testing their absorption and photoluminescence (PL) spectra in PBS (pH = 7.4). Both ASO and SON had a strong absorption peak at 585 nm in PBS solution (Figure 2a) with the high molar absorption coefficients of 5.82×104 and 5.35×104 M-1 cm-1, respectively (Figures S6&S7, Supporting Information). The spectral profiles are 8


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similar to their precursor compounds 4 and 5 (Figure S8, Supporting Information). However, the vibronic band shoulder of ASO at 670 nm was weaker than that of SON (Figure 2a), implying that the π-conjugated segments of ASO was less aggregated as compared with that for SON.43 This should be attributed to the good water-solubility of ASO provided by the long PEG side chains. PL spectra also showed difference between the two nanoparticles. SON had the stronger PL intensity, which was ~2.2-fold higher than ASO in PBS (pH = 7.4) (Figures 2b&c). In fact, SON had the higher quantum yield (0.18%) than that of ASO (0.12%). This phenomenon indicated that the fluorescence intensity was affected not only by the aggregation states of chromophores, but also by surrounding environment. As the backbone of both ASO and SON has the charge transfer characteristic, their excited states are sensitive to the polarity of the environment. With the better water-solubility, the backbone of ASO should expose to water molecules more than that for SON, resulting in increased nonradiative decay and thus quenched fluorescence. To further validate the practicability for optical imaging, the photostability of ASO was studied by tracking the change of PL intensity at 780 nm under excitation at 600 nm and monitoring the absorbance at 680 nm under light irradiation at 680 nm for 30 min (Figure 2e). Results showed that both emission and absorption of ASO remained nearly unchanged. Besides, ASO also had good cytocompatibility as shown by the cell viability assay (Figure 2f). These data confirm that ASO is promising for long-term imaging applications.

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Figure 2. The optical properties of ASO and SON. Absorption (a) and normalized fluorescence spectra (b) of ASO and SON in PBS (pH = 7.4). The fluorescence spectra were recorded with the excitation at 600 nm. Quantified fluorescence intensities (c) calculated from the IVIS fluorescence images (d) of PBS, SON and ASO in PBS. Images were acquired by IVIS living imaging system with the excitation and emission at 600 and at 790 nm, respectively. (e) Photostability test of ASO. Black line: the absorption fluctuation of ASO at 680 nm under 680 nm laser irradiation for 30 min. The power density of laser was fixed at 1 W cm-2. Red line: the fluorescence intensity fluctuation of ASO at 780 nm under 600 nm laser irradiation for 30 min. The power density of laser was fixed at 1 W cm-2. (f) Cell viability of 3T3 fibroblasts after incubation with ASO at different concentrations. The error bars represent the standard deviation of three separate measurements.

The PA intensities of ASO and SON were compared at 680 nm at the same optical concentration, showing that ASO was ~1.8-fold brighter than SON (Figures 3a&b). This was consistent with the quenched fluorescence of ASO, which released light energy more in thermal deactivation upon light excitation.31 With better PA brightness 10


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and higher stability in aqueous solution, ASO should be more promising for imaging application. Thus, the fluorescence and PA signals of ASO were further qualified against different concentrations. Good linearity between the fluorescence intensities and the concentrations was detected (Figures 3c&d). Similarly, the PA signals increased linearly with increased concentrations (Figures 3e&f). These data demonstrate the probability of correlating both fluorescence and PA signals with the concentration of ASO.

Figure 3. PA images (a) and intensities (b) of SON and ASO at 680 nm in PBS solution (pH = 7.4) at the same optical concentration. IVIS fluorescence images (c) and quantified intensities (d) of ASO at various concentrations. The red line represents linear fitting. Images were acquired in IVIS living imaging system with the excitation and emission at 600 and 790 nm, respectively. PA images (e) and quantified intensities (f) of ASO at the concentrations ranging from 0.25 to 4.0 mg mL-1. The red line represents liner fitting. The PA signals were recorded at 680 nm. The error bars represent the standard deviation of three separate measurements. The ability of ASO for in vivo fluorescence and PA dual-modal imaging was validated using xenograft 4T1 tumor mouse model. Before systemic administration of 11



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ASO, the tumor exhibited weak PA signals at 680 nm due to the intrinsic absorption of oxy- and deoxy-hemoglobin in the NIR region (Figure 4c). In contrast, almost no fluorescence signals can be detected at 0 h post-injection as oxy- and deoxy-hemoglobin had no fluorescence (Figure 4a). After tail vein injection of ASO, both the fluorescence and PA intensities in the tumor area gradually increased over time (Figure 4d), which indicated that ASO passively targeted to the tumor through enhanced permeability and retention (EPR) effect due to its small size. Both PA and fluorescence signals in the tumor area reached the maximum at 8 h post-injection (Figure 4d). At this time point, the PA and fluorescence intensities were 2.98- and 26.73-fold higher than that of the tumor background, respectively. The observed higher signal-to-background ratio of fluorescence imaging relative to PA imaging should be due to the higher sensitivity of fluorescence and strong PA background of tumor at 680 nm from hemoglobin. However, after post-injection for 8 h, the fluorescence signals slight decreased while the PA signals dramatically dropped. This should be caused by the difference in the tissue penetration for PA and fluorescence imaging. The tissue penetration depths of PA and fluorescence imaging are at centimeter and millimeter scales, respectively.1 Thus, PA imaging can measure the signals from the nanoparticles accumulated deep into tumor, while fluorescence imaging can only measure the signals from the nanoparticles in the skin and the superficial area of the tumor. As a result, PA imaging is a more precise imaging modality to indicate the clearance from the nanoparticles from the tumor over time.

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The real-time PA spectrum (ranging from 680 to 860 nm) extracted from the tumor of ASO-treated mouse at 8 h post-injection was recorded. This spectrum was different from that of saline-treated mouse but similar to the absorption spectrum of ASO in PBS (Figure 4e vs Figure 2a). Besides, the PA intensity at 680 nm from the tumor of ASO-treated mice was ~3-fold higher than that for saline-treated mice, consistent with the PA data in Figure 4d. These results demonstrated the effective accumulation of ASO in tumor regions that led to the enhancement of PA signals over time. The ex vivo biodistribution of ASO in different organs was obtained by both fluorescence and PA at the post-injection time of 24 h (Figures 4b&f). The tumor signals were very close to that of liver, indicating the good biodistribution of ASO. This should stem from its appropriate size and high structural stability that avoided the particle dissociation, allowing for passive targeting of the tumor in living mice.

Figure 4. PA and fluorescence imaging in vivo. (a) Fluorescence images of a subcutaneous 4T1 tumor in a nude mouse 0 h, 8 h and 24 h after intravenous administration of the ASO. (b) Ex vivo fluorescence imaging of major organs of mice 24 h after systemic administration of ASO. (c) Representative PA maximum imaging 13



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projection (MIP) images of the tumor region from a ASO-treated living mouse at the post-injection time of 0 h, 8 h and 24 h. (d) PA and fluorescence intensities of the tumor area from ASO systemically treated mice at different post-injection time points. (e) In vivo real-time PA spectra extracted from the tumors in living mice after systemic administration of ASO or saline for 8 h. (f) Ex vivo PA and fluorescence quantification of major organs of mice 24 h after systemic administration of the ASO. All the IVIS fluorescence images were obtained with excitation at 600 nm and emission at 790 nm. The PA data were acquired at 680 nm. Error bars represent standard deviations of three separate measurements.

CONCLUSIONS In summary, a NIR-absorbing ASO containing a hydrophobic semiconducting oligomer backbone and hydrophilic PEG side chains was synthesized, fully characterized, and applied for PA and fluorescence dual-modal imaging of tumor in living mice. Due to the amphiphilic feature of ASO, homogenous nanoparticles can be spontaneously constructed via self-assembly in aqueous solution with a relatively small size (~23.84 nm). As a result of the single composition that avoids the micellar dissociation, ASO exhibited superior structural stability over its counterpart nanoparticles (SON) synthesized via nanoprecipitation. Although the better water-solubility of ASO resulted in reduced fluorescence relative to SON, it led to higher PA brightness for ASO due to the increased thermal generation. The small size, high structural stability, photostability and low cytotoxicity of ASO allowed it for tumor imaging in living mice. With its ability to passively accumulate to the tumor tissue through EPR effect, ASO permitted clear visualization of tumor in living mice 14


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with the signal-to-background ratios of ~3 and ~27 for PA and fluorescence imaging, respectively. Our work thus provides non-dissociated small NIR-absorbing organic nanoparticles for in vivo PA and fluorescence dual-modal imaging. In view of the amphiphilicity of such nanoparticles, other hydrophobic components such as optically active molecules or drugs can be encapsulated for other imaging and therapy applications. EXPERIMENTAL SECTION Materials

and

Characterizations.

N,N-Diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline

and

2,5-Bis(6-bromohexyl)-3,6-bis(5-bromothiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5 H)-dione were purchased from Luminescence Technology Corp. (Lumtec). Alkyne-functionalized PEG (95%, average M.W. 5000) was purchased from J&K Scientific Ltd. All the other reagents were purchased from Sigma-Aldrich and used without further purification. NMR data were recorded on a Bruker Ultra Shield Plus 400 MHz NMR, THF-d8 and CDCl3 were used as the solvent. MALDI-TOF-MASS was conducted on a Bruker autoflex under the reflector mode for data acquisition. TEM images were obtained using a JEOL JEM-2100 transmission electron microscope operating at an acceleration voltage of 100 kV. Nanoparticle solutions were dropped onto Formvar-graphite-coated copper grids (300 mesh, Electron Microscopy Science) and air-dried for TEM imaging. DLS was performed on a 90 Plus particle size analyzer (Brookhaven Instruments). UV-visible absorption spectra 15



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were recorded on a Shimadzu UV-3600 UV-VIS-NIR spectrophotometer. PL spectra were measured using a Fluorolog 3-TCSPC spectrofluorometer (Horiba Jobin Yvon). Fluorescence images were obtained by IVIS spectrum imaging system. The methyl thiazolyl tetrazolium (MTT) assay was performed by a PowerWave XS/XS2 microplate spectrophotometer (BioTek, Winooski, VT). A commercial Endra Nexus128 PA tomography system (Endra Inc., Ann Arbor, Michigan) equipped with a tunable nanosecond pulsed laser (680-950 nm, 5 ns pulses, 20 Hz pulse repetition frequency) was used for in vitro or in vivo PA imaging study.

Synthesis

of

2,5-Bis(6-bromohexyl)-3,6-bis(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-2,5-di hydropyrrolo[3,4-c]pyrrole-1,4-dione

(3).

N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1, 0.1 g, 0.27 mmol), 2,5-bis(6-bromohexyl)-3,6-bis(5-bromothiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrro le-1,4-dione

(2,

DPP-2Br,

96.5

mg,

0.123

mmol),

and

palladium-tetrakis(triphenylphosphine) (Pd(PPh3)4) (14 mg) were mixed in a 50 mL schlenk tube. Then the mixture of toluene (10 mL) and potassium carbonate aqueous solution (2M, 3 mL) were added into the schlenk tube, which was degassed by three-freeze-pump-thaw circles. Then the mixture was vigorously stirred at 100 oC for 24 h. After the solvent was removed under reduced pressure, the residue was purified by chromatography using petroleum ether/CH2Cl2 = 1:2 to get the product as a brown 16


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solid (Yield: 112.2 mg, 82%). 1H NMR (400 MHz, THF-d8, ppm) δ: 9.15 (d, 2H), 7.64 (d, 4H), 7.53 (d, 2H), 7.28 (t, 8H), 7.14-7.03 (m, 16H), 4.17 (t, 4H), 3.42 (t, 4H), 1.90-1.76 (m, 8H), 1.29 (s, 8H). MALDI-TOF, m/z: Calcd, 1112.22; Found, 1111.987 [M]+. Synthesis

of

2,5-Bis(6-azidohexyl)-3,6-bis(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-2,5-dihy dropyrrolo[3,4-c]pyrrole-1,4-dione (4). Sodium azide (58.5 mg, 0.9 mmol) was added to a solution of compound 3 (100 mg, 0.0899 mmol) in mixed solvent containing anhydrous DMF (15 mL) and THF (15 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 organic layer was concentrated and purified by chromatography using petroleum ether/CH2Cl2 = 1:3 to get the product as a brown solid (86.6 mg, 93%). 1H NMR (400 MHz, THF-d8, ppm) δ: 9.15 (d, 2H), 7.62 (d, 4H), 7.53 (d, 2H), 7.32-7.23 (m, 8H), 7.14-7.00 (m, 16H), 4.16 (t, 4H), 3.27 (t, 4H), 1.83-1.75 (m, 4H), 1.64-1.54 (m, 4H), 1.48 (s, 6H), 1.29 (s, 4H). MALDI-TOF, m/z: Calcd, 1036.40; Found, 1036.201 [M]+. Synthesis of ASO (5). The final product ASO (5) was synthesized via “click” reaction. Compound 4 (80 mg, 0.0772 mmol), mPEG-Alkyne (Mw 5000, 965 mg, 0.193

mmol),

CuBr

(55.6

mg,

0.386

mmol)

and

N,

N,

N',

N'',

N''-Pentamethyldiethylenetriamine (PMDETA, 506 µL) were mixed in a 50 mL round-bottom flask, which was degassed and purged with N2. Then 20 mL of THF was injected into the flask and the mixture was stirred at room temperature for 3 days. After the solvent was removed under reduced pressure, the mixture was reconstituted 17



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into excess deionized water under ultrasonic conditions. Subsequently, the resulting solution was purified by dialysis against pure water for 3 days with a dialysis bag with the molecular weight cut-off of 50 kDa to remove the small molecules. Finally, the solution was extruded using a manual extruder through 0.22 µm membranes, and freeze-dried to obtain blue floccules (626.7 mg, 73.6%). 1H NMR (400 MHz, CDCl3, ppm) δ: 8.89 (s, 2H), 7.61-6.82 (m, 30H), 4.20-4.00 (m, 4H), 4.05 (m, 4H), 3.82-3.40 (m, 1048H), 3.37 (s, 6H), 1.50-0.75 (chemical shifts of the proton in alkyl chains). MALDI-TOF MS (the maximum peak, m/z): 11029.39 (M+). Preparation of Nanoparticles. ASO nanoparticles were prepared by directly dissolving ASO in water under continuous sonication. SON was synthesized by a nanoprecipitation method. In brief, compound 4 (1.0 mg) and DSPE-mPEG5000 (20 mg) were dissolved in 1.5 mL of THF, which was rapidly injected into a mixture of water (9 mL) and THF (1 mL) under continuous sonication for 1 min. THF was then removed by argon blowing on the solution surface under stirring at room temperature. The resulting nanoparticles were purified by filtration through a 0.22 µm PVDF syringe driven filter (Millipore) and stored at 4 oC for further use. Cytotoxicity Test. MTT assays were performed to evaluate the cytotoxicity of the ASO using 3T3 fibroblasts. The cells were seeded in 96-well plates (Costar, Chicago, IL) at an intensity of 2×104 cells mL-1. After 24 h of incubation, the medium was replaced by the ASO solution at the concentration of 10, 20, 40, 60, and 80 µg mL-1, and the cells were incubated for another 24 h. After the designated time intervals, the wells were washed twice with PBS buffer, and 100 µL MTT solution (0.5 mg mL-1) 18


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was added to each well. After incubation for 3 h at 37 ºC, the MTT medium solution was carefully removed and the formazan crystals were solubilized with 200 µL of dimethylsulfoxide (DMSO) for 15 min. The absorbance value was recorded at 490 nm using a microplate reader. The absorbance of the untreated cells was used as a control with its absorbance as the reference value for calculating 100% cellular viability. Tumor Mouse Model. The 4T1 tumor bearing nude mice were purchased from Nanjing Mergene Life Science Co., Ltd. and used according to the guideline of the Laboratory Animal Center of Nanjing Mergene Life Science Co., Ltd. To establish tumor models in six-week-old female nude mice, two million 4T1 cells suspended in 50 mL of 50% v/v mixture of Matrigel in supplemented DMEM (10% fetal bovine serum, 1% pen/strep (100 IU mL-1 penicillin and 100 µg mL-1 streptomycin) were injected subcutaneously in the wanted region of the mouse. Tumors were grown until a single aspect was ~10 mm before used for in vivo imaging experiments. Fluorescence Imaging In Vivo. 4T1 tumor xenografted nude mice were anesthetized using 2% isoflurane in oxygen, ASO was systematically injected through the tail vein using a microsyringe. Fluorescence imaging was performed using IVIS spectrum imaging system. Fluorescence images of the mice were acquired at designated time points after ASO administration. The first and last fluorescence images were recorded 0.5 and 24 h post-injection, respectively. For ex vivo fluorescence imaging, mice were sacrificed by CO2 asphyxiation, and the tumor, liver, spleen, heart, lung, kidney, intestine and muscle were harvested for ex vivo fluorescence imaging to estimate the tissue distribution of the ASO. 19



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PA Imaging In Vivo. 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 nm before systemic administration with the ASO (150 µL, 1.0 mg mL-1) (n = 3) and after different post-injection time periods. Mice treated with saline (150 µL) (n = 3) were set as the control. Data was acquired through a continuous model that took 12 s to obtain one data set. Three dimensional PA image was reconstructed off-line using data acquired from all 128 transducers at each view and a back-projection algorithm. Real-time PA spectra were recorded with a LAZR instrument (Visualsonics, 2100 High-Resolution Imaging System). For ex vivo PA imaging, mice were sacrificed by CO2 asphyxiation, and organs were embedded in agar phantom and acquired immediately with Endra Nexus128 PA imaging system. 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. Fluorescence intensities were measured by region of interest (ROI) analysis using the IVIS living imaging system. Intensities of PA signal were determined by 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., CA, USA).

ASSOCIATED CONTENT

Supporting Information 20


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This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Figures S1-S5 detail the structure characterization of all the intermediates and the final product, ASO (5). Supporting Figure S6 shows the photophysical properties of compound 4 and 5 in THF solution. Supporting Figures S7&S8 detail the absorption performance of ASO and SON, respectively.

AUTHOR INFORMATION

Corresponding Authors *

E-mail: [email protected]

*

E-mail: [email protected]

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS K.P. thanks Nanyang Technological University (Start-Up grant: NTU-SUG: M4081627.120) and Singapore Ministry of Education (Academic Research Fund Tier 1: RG133/15 M4011559 and Academic Research Fund Tier 2 MOE2016-T2-1-098) for the financial support. W.H. and Q.F. thank the National Basic Research Program of China (No. 2012CB933301), the National Natural Science Foundation of China 21



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(No. 21674048, 21574064, 61378081, 11404219, and 61505076), Synergetic Innovation Center for Organic Electronics and Information Displays, and the Natural Science Foundation of Jiangsu Province of China (No. BZ2010043, NY211003, BM2012010) for the financial support.

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Amphiphilic Semiconducting Oligomer for Near-Infrared Photoacoustic

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