Molecular Engineering of Conjugated Polymers for Biocompatible

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Molecular Engineering of Conjugated Polymers for Biocompatible Organic Nanoparticles with Highly Efficient Photoacoustic and Photothermal Performance in Cancer Theranostics Bing Guo,†,⊥ Zonghai Sheng,‡,⊥ Dehong Hu,‡ Anran Li,§ Shidang Xu,† Purnima Naresh Manghnani,† Chengbo Liu,∥ Lin Guo,§ Hairong Zheng,*,‡ and Bin Liu*,† †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore ‡ Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, People’s Republic of China § Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology, Ministry of Education, School of Chemistry, Beihang University, Beijing 100191, People’s Republic of China ∥ Research Laboratory for Biomedical Optics and Molecular Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, People’s Republic of China S Supporting Information *

ABSTRACT: Conjugated polymer nanoparticles (CP NPs) are emerging candidates of “all-in-one” theranostic nanoplatforms with dual photoacoustic imaging (PA) and photothermal therapy (PTT) functions. So far, very limited molecular design guidelines have been developed for achieving CPs with highly efficient PA and PTT performance. Herein, by designing CP1, CP2, and CP3 using different electron acceptors (A) and a planar electron donor (D), we demonstrate how the D−A strength affects their absorption, emission, extinction coefficient, and ultimately PA and PTT performance. The resultant CP NPs have strong PA signals with high photothermal conversion efficiencies and excellent biocompatibility in vitro and in vivo. The CP3 NPs show a high PA signal to background ratio of 47 in U87 tumor-bearing mice, which is superior to other reported PA/PTT theranostic agents. A very small IC50 value of 0.88 μg/mL (CP3 NPs) was obtained for U87 glioma cell ablation under laser irradiation (808 nm, 0.8 W/cm2, 5 min). This study shows that CP NP based theranostic platforms are promising for future personalized nanomedicine. KEYWORDS: conjugated polymer nanoparticles, molecular engineering, theranostic platform, tumor, photoacoustic imaging, photothermal therapy applications. Therefore, it is of great value to develop “all-in-one” theranostic nanoplatform based on a single material that intrinsically contains both diagnosis and therapy functions.6−10 Among different theranostic modalities, light-activatable nanoplatforms with dual photoacousctic imaging (PA) and photothermal

T

heranostic nanoplatforms are able to offer real-time identifying of tumor location, monitoring of drug distribution/accumulation, selective ablating of tumor, and visualizing of therapeutic outcome, which are thus promising to address the unmet medical needs.1−11 The current theranostic nanoplatforms are generally fabricated by covalently or physically combining various diagnostic and therapeutic units together,3−5 which leads to a complicated fabrication process with compromised reproducibility and concerns of unstable nanostructures for in vivo © 2017 American Chemical Society

Received: July 4, 2017 Accepted: September 11, 2017 Published: September 11, 2017 10124

DOI: 10.1021/acsnano.7b04685 ACS Nano 2017, 11, 10124−10134

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Cite This: ACS Nano 2017, 11, 10124-10134

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Figure 1. (a) Chemical structures of CP1−3. (b) Illustration of nanoparticle formation. (c) Representative photo of CP3 NP suspension. (d) TEM image of CP3 NPs. (e) Representative DLS result of CP3 NPs.

fluorescence in NPs.33−35 Most of these polymers, however, do not show an optimized light absorption coefficient at the most widely used PTT excitation wavelength of 808 nm.33−35 Recently, we have developed donor−acceptor (D−A) CPs with strong intramolecular charge transfer (ICT) characteristics, and these CP NPs showed moderate molar absorptivity at 808 nm, with intrinsically low fluorescence, inhibited phototoxicity, and promising PTT/PA performances.36−38 As the light harvesting property is directly related to the extinction coefficient of CP NPs, which greatly affects their PTT/PA performance, it is therefore ideal if one can develop a feasible molecular engineering strategy to tailor the CP structures for large molar absorptivity and high PA/PTT performance. In this contribution, we demonstrate polymer molecular engineering on how to build D−A CP NPs with good PA and PTT performance for effective cancer theranostics. The molecular engineering of a CP is based on the selection of a planar donor and electron-deficient acceptors (Figure 1a) to build up polymeric backbones, since rigid planar structures are highly beneficial for large extinction coefficients of CPs.39,40 More importantly, the higher D−A strength could introduce stronger ICT and thus narrower band gap, leading to red-shifted absorption. To demonstrate the concept, we started with the polymer synthesis and characterization. After CP NP formation, their PA imaging and PTT in vitro and in vivo were evaluated. The developed CP NPs exhibit superior performance to many reported PA/PTT agents, including ICG, one of the most popular PA/PTT agents.

therapy (PTT) functionalities exhibit advantages over conventional theranostic nanoplatforms (e.g., fluorescence-guided chemotherapy). PA agents naturally possess photothermal functions, and they can synergistically offer deep penetration, high spatial resolution, and effective therapeutic function with minimal side-effects.8−16 Efficient exogenous PA/PTT agents are desirable to have the following characteristics: (i) biocompatibility, (ii) strong NIR absorption (λ = 700−1100 nm, preferably at 808 nm to match the common PTT laser excitation) for efficient light harvesting and deep tissue penetration; (iii) excellent photostability, (iv) favorable nonradiative exciton decay pathway to efficiently produce photothermal energy, (v) good thermal conductivity and effective generation of acoustic waves, and (vi) ease to process into nanoparticles (NPs) for passive/active tumor-targeting.8−16 So far, several types of PA/PTT agents have been reported in the literature.11−16 Inorganic PA/PTT agents (e.g., gold,17 carbon materials,7,18 semiconductor nanoparticles,19 quantum dots,8 and tungsten nanoparticles) have been widely investigated, which face the challenges of long-term safety.11−16,20 Small organic molecules (e.g., indocyanine green (ICG),21−24 methylene blue,25 perylene-piimide,26 terrylenediimide,27 diketopyrrolopyrrole-triphenylamine,28 and porphyrin) have the advantage of fast body clearance.29 However, they usually show different degrees of fluorescence and reactive oxygen species generation, which consume exciton energy and thus lead to moderate PA/PTT efficacy and fast photodegradation.21−25,28,29 Despite the disadvantages, the FDA-approved ICG remains one of the most widely investigated PA/PTT agents because of its high extinction coefficient and low fluorescence quantum yield in aqueous media.21−24 Conjugated polymers (CPs) have recently emerged as one of the most promising candidates for PA/PTT applications.12,13,30,31 It has been reported that CPs are able to provide much higher PA signal than gold nanorods and carbon nanotubes based on the same mass.32 So far, several strategies have also been introduced to amplify the PA/PTT efficacy, which include the introduction of electron quenchers or energy acceptors to quench the CP

RESULTS AND DISCUSSION Synthesis of Conjugated Polymers CP1−3 and the NP Formation. The polymer synthesis was started from 4H-dithieno[3,2-b:2′,3′-d]pyrrole (DTP),41,42 which was copolymerized with acceptors including benzothiadiazole (BT), pyridal[2,1,3]thiadiazole (PT), and diketopyrrolopyrrole (DPP), via Stille coupling reaction to yield CP1−3 (Figure 1a, Schemes S1−3). The D−A strength sequence of CPs follows 10125


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Figure 2. Optimized confirmation for CP1−3 trimer structures and illustration of the torsion angles along the conjugated backbones.

Figure 3. (a−c) HOMO and LUMO wave functions of the CP1−3 trimer with the geometry-optimized structures (B3LYP/6-31G(d,p)).

conformation (Figure 2 and Figure S2), indicating coplanarization of D−A repeating units along the backbones. Although DPP is bulkier and stiffer than both BT and PT, CP3 still shows quite a planar structure due to the adjacent bisthiophene rings, which can effectively reduce the torsion angle.42 The coplanarity favors extended conjugation length through the backbones, which offers large extinction coefficients for CP1−3. The planar backbone structure also facilitates strong intra/interchain electronic interaction in solution/solid states,44,45 to yield long-wavelength absorption, which is consistent with the UV−vis absorption spectra. As shown in Figures 3 and S3, the HOMO wave functions of CP1−3 are well delocalized along the backbones, while the LUMO wave functions are more localized on the electrondeficient acceptors, indicating effective ICT within these molecules. Furthermore, the calculated result also shows that CP3 has the strongest oscillator strength, which agrees with its high extinction coefficient (Figure S1c). Characterization of Photophysical Properties of CP NPs. Subsequently, CP1−3 were transformed into wasterdispersible NPs through nanoprecipitation using 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG2000) as the matrix (Figure S1).31 The obtained NPs (Figure 1c) show spherical shapes with sizes of

CP1 < CP2 < CP3. CP1−3 dissolve well in organic solvents such as tetrahydrofuran (THF) and chloroform. In THF, all three CPs show two absorption peaks. The ones at shorter wavelengths of 413 nm for CP1, 415 nm for CP2, and 420 nm for CP3 are derived from π−π* transition of the polymer backbone. Meanwhile, the peaks at the longer wavelengths are due to ICT, which occur at 703 nm for CP1, 748 nm for CP2, and 858 nm for CP3 with mass extinction coefficients of 55.0, 62.8, and 95.7 L g−1 cm−1, respectively (Figure S1a). Additionally, CP1−3 show very weak emission peaks at 866, 865, and 885 nm with fluorescence quantum yields of 3.2‰, 0.74‰, and 0.22‰, respectively (Figure S1b). It is obvious that, for CP1−3, the higher D−A strength leads to stronger ICT, more red-shifted absorption, and lower emission intensity in aqueous media.43 To understand the optical properties of CP1−3, both the density functional theory (DFT) and the time-dependent DFT (TDDFT) calculations were conducted via the Gaussian 09 program using a trimer model to investigate their optimized geometry, wave functions, and oscillator strength.39 The solubilizing side-chains attached to the conjugated backbones were represented with methyl groups to minimize the computational time. The optimized trimer structures of CP1−3 exhibit planar structures with small dihedral angles in both all-trans and all-cis 10126


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Figure 4. Study of light harvesting and exciton energy dissipation of CP NPs (0.0125 mg/mL). (a) UV−vis spectra. (b) Normalized photoluminescence (PL) spectra. (c) Fluorescence images. (d) Degradation of 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) in the presence of NPs under NIR laser irradiation (0.8 W/cm2) for different times. (e) Temperature monitoring of CP NPs under NIR laser illumination (808 nm, 0.8 W/cm2). (f) NIR thermal images of CP NPs under NIR laser illumination (808 nm, 0.8 W/cm2, 3 min).

Figure 5. In vitro PA study. (a) PA spectra of CP NPs and ICG (0.125 mg/mL). (b) PA intensity of CP NPs and ICG (0.125 mg/mL) excited at 780 nm. (c) PA images of CP NPs and ICG (0.125 mg/mL) excited at 780 nm. (d) PA signal of CP3 NPs at different concentrations. (e) PA amplitudes of CP3 NPs at 780 nm as a function of concentration. (f) PA amplitudes of CP3 NPs at 780 nm under exposure to different numbers of laser pulses.

about 43 to 52 nm by transmission electron microscopy (TEM), respectively (Figure 1d and Figure S4). The NPs of CP1−3 in water exhibited broadened absorption peaks at 690, 740, and 783 nm with mass extinction coefficients of 41.4, 44.0, and 59.3 L g−1 cm−1, respectively (Figure 4a). As compared to that for polymer molecules in THF, the decreased extinction coefficient for CP NPs is due to the attenuated ICT effect and interamolecular and intramolecular π−π stacking of the CPs in the DSPE-PEG matrix. Planar structures favor inter/intrachain packing and strong π−π interactions, which therefore lead to slightly broadened absorption spectra for NPs. Importantly, CP3 NPs showed more blue-shifted and broadened absorption, as compared to CP1,2, indicating that the stiff structure leads to more intensified inter/intrachain interactions. Due to the broad

absorption of CP3, the extinction coefficient of CP3 NPs at 808 nm is 57.7 L g−1 cm−1, which is very similar to that at 783 nm (Figure 4a). The extinction coefficient of CP3 NPs at 808 nm is larger than many reported PA/PTT agents including Cu2−xSe NPs (8.5 L g−1 cm−1),19 Bi2Se3 nanosheet (11.5 L g−1 cm−1),46 PorCP (34.7 L g−1 cm−1),36 PFTTQ (3.6 L g−1 cm−1),37,38 and ICG (47.0 L g−1 cm−1) (Figure S5a), demonstrating the strong capabilities of the CP NPs in light harvesting. The photoluminescence (PL) spectra of NPs in water are shown in Figure 4b. At the same mass concentration, CP1 NPs show the brightest emission, which is followed by CP2 and CP3 NPs. All the CP NPs are less emissive than ICG (Figure S5b). Furthermore, these CP NPs do not produce obvious reactive oxygen species under continuous 808 nm laser irradiation 10127


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Figure 6. Cytotoxicity evaluation of CP3 NPs. (a) Viabilities of U87 glioma cells and (b) bEnd.3 endothelial cells against CP3 NP concentrations. (c) Fluorescence images of U87 glioma cells treated with 2 μg/mL of CP3 NPs and NIR laser irradiation (808 nm, 0.8 W/cm2, 5 min), in which live and dead U87 glioma cells were costained with calcein-AM (with green emission) and propidium iodide (with red emission), respectively. (d) Viabilities of U87 glioma cells under NIR laser treatment (808 nm, 0.8W/cm2, 5 min).

NPs. Considering that at 780 nm the CP2 and CP3 NPs only show 1.6- and 2.2-fold higher extinction coefficients than CP1 NPs, the higher PA signals for CP2 and CP3 NPs indicate that the quenched emission contributes to the higher PA signals. Notably, ICG as a reference shows much lower PA amplitude than CP NPs at the same mass concentration. This is partially due to the fact that ICG also shows substantial fluorescence emission (Figure S5b) and ROS generation,21 which compete with the photothermal conversion. Further in vitro phantom studies reveal linear correlation between the mass concentration and the PA signals for CP3 NPs (Figure 5d,e), suggesting possible PA intensity quantification of NPs in vitro and in vivo.48 Moreover, the PA intensity of CP3 NPs is almost constant even after 5000 laser pulse irradiations (Figure 5f), which is highly desirable for PA imaging. Cytotoxicity and in Vitro PTT of CP3 NPs. Given the good PA/PTT performance as well as the excellent photostability of CP3 NPs, the cytotoxicity and PTT performance were further examined with a standard CCK-8 assay. More than 90% viability was maintained after incubation of U87 glioma and bEnd.3 cells with CP3 NPs, respectively, even at an NP concentration as high as 100 μg/mL, indicating good biocompatibility of CP3 NPs (Figure 6a,b). Under 808 nm NIR laser irradiation, the CP3 NPs in glioma cells did induce local hyperthermia to kill cancer cells. The results were visually evaluated by live/dead staining, in which green-emissive calcein-AM and red-emissive propidium iodide stained live and dead cells, respectively (Figure 6c). In all control groups, cells exhibited obvious green fluorescence, suggesting that NPs are biocompatible and laser irradiation alone causes negligible cancer cell apoptosis. In the PTT-treatment group, the U87 glioma cells showed strong red fluorescence, revealing that CP3 NPs under NIR irradiation could efficiently ablate U87 cancer cells. A quantitative assay was further performed with CCK-8 analysis (Figure 6d). It was observed that neither NPs nor NIR irradiation alone could induce obvious cell ablation. In contrast, NPs and NIR irradiation could greatly inhibit

(Figure 4d and Figure S6). Both the low emission and negligible reactive oxygen species (ROS) production favor CP1−3 NPs for high photothermal effect.47 The photothermal effect not only can be applied to destruct tissues in vivo for PTT but also can be used to induce elastic expansion accompanied by the generation of acoustic waves for PA imaging.11−16 PA and PTT Properties of CP1−3 NPs. To study the photothermal effect, an infrared thermalmeter was used to quantitatively monitor the temperature change of CP NPs under continuous laser irradiation. It is noticed that after 3 min of 808 nm laser irradiation (0.8 W/cm2) the CP3 NP temperature rapidly increased to 68.0 °C, while the temperature of CP1,2 NPs moderately approached 56.0 and 61.2 °C, respectively (Figure 4e,f). The photothermal effect of CP3 NPs was further studied at different NP concentrations (Figures 4e and S7a). The photothermal temperature of the CP3 NP suspension increases with the NP concentration, which approaches a plateau temperature of 77 °C within 7 min. The photothermal stability of CP3 NPs was investigated by (i) absorption and morphology changes before and after irradiation and (ii) cyclic photothermal heating and cooling processes. As compared to fresh CP3 NPs, 1 h of irradiation with an 808 nm laser (0.8 W/cm2) does not cause any obvious change in their absorbance or morphology (Figures S7b, S8, and S9). The cyclic photothermal heating and cooling processes also revealed that the NPs can be reversibly photothermally heated for five cycles without any noticeable change (Figure S8c). Under the same conditions, ICG can only reach a moderate temperature of around 34 °C (Figure S5c). The poor photothermal performance of ICG is mainly attributed to its severe photobleaching, which is confirmed by the absorbance loss after 5 min of continuous NIR laser irradiation (Figure S5d). Encouraged by the good photophysical properties of CP1−3 NPs, their PA spectra were tested at the same mass concentration (Figure 5a−c). Upon excitation at 780 nm, the CP2 and CP3 NPs produced 2.8- and 5.8-fold brighter PA signals than CP1 10128


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at 24 h postinjection, the PA signal in tumor shows a very high signal to background ratio (S/B) of 47 at a depth to 3.2 mm, which is superior to many PA contrast agents (Table S2). Ex vivo PA image of the excised tumor tissue further revealed that the PA signal at 24 h postinjection was derived from NPs in tumor (Figure 7b). For comparison, the in vivo tumor imaging was also performed with ICG at the same mass concentration as CP3 NPs. As shown in Figure S10, at 1 h postinjection of ICG, the PA signal in tumor reached the maximal contrast with an S/B of 5, and the ICG can only image the superficial tumor surface. These results revealed that CP3 NPs are superior to ICG in PA imaging. In Vivo PTT of CP3 NPs. Encouraged by the efficient PTT performance in vitro and high PA contrast in vivo, the PTT efficacy of CP3 NPs was examined in vivo on a subcutaneous U87 glioma-bearing mice model (Figure 8 and Figure S11). According to the PA imaging results, at 24 h post CP3 NP injection, the NPs reached high accumulation and penetrated into the deep tumor region. As a consequence, the mice were treated with localized laser irradiation on tumor at 24 h postinjection. Upon laser irradiation, the tumor temperature of the mice treated with CP3 NPs rapidly jumped to 52.7 °C within 5 min, while the control group exhibited only a slight temperature increase to 34.5 °C after the same exposure period (Figure 8a). The temperature images further confirmed the accumulation and strong photothermal conversion ability of CP3 NPs in tumor. Furthermore, hematoxylin and eosin (H&E) staining analysis after laser treatment revealed that the local hyperthermia can severely damage tumor tissue via cell necrosis, while neither NPs alone nor laser irradiation alone affected tumor growth (Figure 8e). These results confirm that the CP3 NPs are capable of mediating effective photothermal destruction of solid tumors. To quantitatively evaluate the PTT efficiency, the tumor growth rate was continuously recorded every 3 days after treatments (Figure 8b−d). The control groups showed rapid tumor

Table 1. PTT Conditions to Achieve More Than 50% of U87 Cells Ablation in Vitro PTT agents

concentration μg/mL

808 nm laser

Cu2‑xSe NPs20 CuS-ferritin nanocages49 nano graphene oxide50 porphyrin-graphene oxide51 ICG NPs52 polypyrrole microspheres53 CP3 NPs

10 10 6600 100 10 5 0.88

0.8 W/cm2, 5 min 0.8 W/cm2, 5 min 15.3 W/cm2, 8 min 0.8 W/cm2, 4 min 1.25 W/cm2, 5 min 6 W/cm2, 5 min 0.8 W/cm2, 5 min

indicating great potentials of CP3 NPs for in vivo cancer cell ablation. In Vivo PA Imaging of CP3 NPs. Encouraged by the good PA performance of CP3 NPs in vitro, the in vivo PA imaging was validated with a subcutaneous U87 xenograft mouse model. Dual-modal PA and ultrasound images of the tumor crosssection were synchronously monitored with a homemade AR-PAM system (Supporting Information). While ultrasound imaging was used to visualize tumor boundaries, PA was applied to investigate the NP distribution and resultant PA contrast variation in tumors. As shown in Figure 7, before intravenous injection of CP3 NPs, a weak PA signal derived from hemoglobin endogenous contrast in blood vessels was observed under skin or in tumors upon excitation at 780 nm. After injection of CP3 NPs (0.5 mg/kg), the PA contrast in tumor rapidly increased. More importantly, the deeper tumor region was clearly imaged over time, indicating that the NPs were able to accumulate in tumor tissues via enhanced permeability and retention effect. Notably,

Figure 7. In vivo PA study of CP3 NPs (injection dose = 0.5 mg/kg). (a) In vivo PA images of CP3 NPs in subcutaneous U87 xenograft tumor model. (b) Photos of PA of exfoliated tumor at 24 h postinjection with CP3 NPs. (c) Statistical results of PA signal in the tumor region at different times postinjection of CP3 NPs. 10129


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Figure 8. In vivo PTT of mice bearing subcutaneous U87 xenograft tumors which were divided into four groups including PBS group, NPs group, laser group, and laser + NPs group. (a) Infrared thermal images of mice under NIR laser irradiation (808 nm, 0.8 W/cm2). (b) Tumor growth curves. (c) Survival curves. (d) Mice body weight curves. (e) H&E stained images of tumor sections from mice after 4 h of PTT treatment (magnification: 400×). (f) Representative photos of mice.

Figure 9. Hematology and blood biochemical assay of mice at 7 d postinjection of CP3 NPs. Results and standard deviation of (a) mean corpuscular hemoglobin concentration (MCHC); (b) concentration of hemoglobin (HGB); (c) hematocrit (HCT); (d) neutrophilic granulocytes number (Gran); (e) number of white blood cells (WBC); (f) number of red blood cells (RBC); (g) mean corpuscular volume (MCV); (h) mean platelet volume (MPV); and (i) platelets (PLT).

growth with 0% survival rate on day 21, suggesting that neither NP injection alone nor laser treatment alone can inhibit tumor growth. In contrast, for the CP3 NP-mediated PTT treatment group, the growth of U87 tumor was totally eliminated and no tumor recurrence was found, with a survival rate of 100% on day 30. Moreover, during the PTT treatment process, neither significant body weight loss nor an obvious body weight difference

was observed among all groups, indicating insignificant sideeffects of the treatment process. The excellent biocompatibility and high tumor ablation efficiency via PTT in vivo are consistent with the results in vitro. In Vivo Toxicity Evaluation of CP3 NPs. A general concern of nanomaterials for potential clinical applications is their toxicity in vivo. CP3 NPs were investigated with dual blood 10130


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polymer solution was carefully concentrated via rotary evaporator and was washed with excess methanol. The precipitant was dried under vacuum to yield CP1 as a dark solid (52 mg, yield 65%). 1H NMR (500 MHz, C2D2Cl4, 100 °C): δ 8.30−7.78 (m, 2H), 7.25−7.02 (m, 2H), 4.26−4.09 (m, 2H), 2.09 (br, 1H), 1.60−1.31 (m, 24H), 0.91(br, 6H). GPC (THF, polystyrene standard), Mn: 1.73 × 104 g/mol; Mw: 2.47 × 104 g/mol, PDI: 1.4. Synthesis of CP2. A solution of 4,7-dibromo[1,2,5]thiadiazolo[3,4c]pyridine (44.24 mg, 0.15 mmol), 4-(2-hexyldecyl)-2,6-bis(trimethylstannyl)-4H-dithieno[3,2-b:2′,3′-d]pyrrole (109.4 mg, 0.15 mmol), Pd2(dba)3 (2.1 mg, 2.25 μmol), and P(o-tol)3 (2.7 mg, 9.00 μmol) in toluene (8 mL) was stirred at 90 °C under argon for 2 days. After the reaction was completed, the polymer was purified following the same protocol with CP1. CP2 was collected as a dark solid (43 mg, yield 53%). 1H NMR (500 MHz, C2D2Cl4, 100 °C): δ 8.81−6.81 (m, 3H), 4.11 (br, 2H), 2.13 (br, 1H), 1.48−1.35 (m, 24H), 0.93 (br, 6H). GPC (THF, polystyrene standard), Mn: 9.74 × 103 g/mol; Mw: 2.08 × 104 g/mol, PDI: 2.1. Synthesis of CP3. A solution of 3,6-bis(5-bromothiophen-2-yl)-2,5bis(2-octyldodecyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (71.4 mg, 0.07 mmol), 4-(2-hexyldecyl)-2,6-bis(trimethylstannyl)-4H-dithieno[3,2-b:2′,3′-d]pyrrole (51.1 mg, 0.07 mmol), Pd2(dba)3 (1.0 mg, 1.05 μmol), and P(o-tol)3 (1.1 mg, 3.20 μmol) in toluene (8 mL) was stirred at 90 °C under argon for 2 days. After the reaction was completed, the polymer was purified according to the same procedure with CP1. CP3 was obtained as a dark solid (59 mg, yield 67%). 1H NMR (500 MHz, C2D2Cl4, 100 °C): δ 8.87 (br, 2H), 7.48−7.04 (br, 4H), 4.11 (br, 4H), 2.08 (br, 2H), 1.45−1.32 (m, 54H), 0.93 (br, 12H). GPC (THF, polystyrene standard), Mn: 2.12 × 104 g/mol; Mw: 3.26 × 104 g/mol, PDI: 1.5. Preparation of CP NPs. For the synthesis of CP NPs, conjugated polymer (0.5 mg) and DSPE-PEG2000 (1 mg) were completely dissolved in 1 mL of THF. The resultant solution was further sonicated for 10 min in a water bath at room temperature. The mixture was subsequently injected into 10 mL of DI water. Sonication was immediately applied with a microtip probe sonicator (12 W) for 150 s. After sonication, the mixture was stirred in a fume hood at room temperature for 24 h to remove THF. The NP suspension was further filtered via a 0.2 μm syringe filter. Then NPs were washed several times with DI water and subsequently concentrated to 1 mg/mL with Corning concentrators (Mw 100 000). Photostability Test of CP NPs. To study CP NP photostability, the NP suspension at 0.0125 mg/mL was irradiated with a NIR laser (808 nm, 0.8 W/cm2) for 1 h. The UV−vis absorption spectra and TEM images of the NP suspension before and after NIR irradiation were recorded, respectively. ROS Generation Test. To check whether the CP NPs can generate ROS, the NP suspension at 0.0125 mg/mL was NIR laser irradiated (808 nm, 0.8 W/cm2) using 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) as the indicator. The UV−vis spectra of ABDA were monitored at different durations of NIR laser irradiation. Cell Culture and Animal Model. Human brain glioma cell line U87 cells and microvascular endothelial cell line (bEnd.3) cells were supplied by American Type Culture Collection. They were cultured with Dulbecco’s modified Eagle’s medium (DMEM) media in a humidified environment with 5% CO2 at 37 °C. The DMEM was added with 10% fetal bovine serum and 1% penicillin−streptomycin. Before starting experiments, the cells were precultured until confluence was achieved. All animal studies were conducted according to the animal use and care regulations at Shenzhen Institutes of Advanced Technology. Balb/c nude mice (5−8 weeks old, about 20 g) were supplied by the Medical Experimental Animal Center of Guangdong. To prepare U87 tumorbearing nude mice, U87 cells (3 × 106) in PBS (100 μL) were subcutaneously injected in the nude mice. Once the tumor size increased to about 80 mm3, the mice were used for PA and PTT experiments. Photothermal Cytotoxicity in Vitro. For the cytotoxicity assay, 5 × 104 U87 and bEend.3 cells per well were seeded in a 96-well plate (Costar, IL, USA) and cultured for 24 h, respectively. Then CP3 NPs with different concentrations were added into the 96-well plate for another 24 h. The relative cell viability was detected by the standard

biochemical assay and histology analysis. No significant changes were observed at all doses of biochemical indexes after intravenous injection of CP3 NPs (Figure 9), suggesting that the administration of NPs does not affect normal blood function. The major organs H&E stained images did not show obvious apparent cellular structure changes in 30 days, further indicating low cytotoxicity of the CP3 NPs (Figure S12).

CONCLUSION In summary, we synthesized and systematically studied the photophysical properties and PA and PTT performance of three CPs with D−A structures. With increasing D−A strength, the corresponding CP NPs showed red-shifted absorption, higher extinction coefficients, more inhibited fluorescence emission, faster photothermal heating, and stronger improved photoacoustic signal. The CP NPs demonstrated excellent biocompatibility, good photostability, and highly efficient photothermal therapeutic efficacy in vitro and in vivo, which are superior to many contrast agents, including ICG. Notably, CP NPs also exhibited very high PA contrast with deeper tumor tissue imaging ability. Overall, we demonstrate that CP NPs are efficient PA/PTT agents for theranostic applications. EXPERIMENTAL SECTION Materials. 4,7-Dibromobenzo[c][1,2,5]thiadiazole (BT monomer) was provided by Puyang Huicheng and was purified via recrystallization in methanol. 4-(2-Hexyldecyl)-2,6-bis(trimethylstannyl)-4H-dithieno[3,2-b:2′,3′-d]pyrrole (DTP monomer) and 3,6-bis(5-bromothiophen-2-yl)-2,5-bis(2-octyldodecyl)pyrrolo[3,4-c]pyrrole-1,4 (2H,5H)dione (DPP monomer) were supplied by Solarmer, Beijing, China. 4,7-Dibromo[1,2,5]thiadiazolo[3,4-c]pyridine (PT monomer) was obtained from Derthon, Shenzhen, China. DSPE-PEG2000 was supplied by Avanti Polar Lipids. Tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) and tri(o-tolyl)phosphine (P(o-tol)3) were supplied by Sigma. Toluene, methanol, and tetrahydrofuran were obtained from Fisher. All other chemicals were used as received. Characterization. NMR spectra were characterized with a Bruker 500 NMR spectrometer. In NMR spectra, the chemical shifts of the residual solvent peaks and tetramethylsilane at δ = 0.00 ppm were used as reference. Gel permeation chromatography (GPC) results were tested via a Waters 996 photodiode detector and an aphenogel GPC column. The running conditions are a polystyrene standard in THF eluent at 1.0 mL/min at room temperature. UV−vis spectra were studied with a Shimadzu model UV-1700 spectrometer. PL spectra were tested using a PerkinElmer LS 55 spectrometer. The NP size and its distribution were tested with laser light scattering and field emission transmission electron microscopy, JEOL JEM-2010, respectively. All PA experiments were carried out with a homemade acoustic-resolution PA microscopy system (Supporting Information). The PA spectra of the CP NPs and ICG were tested from 700 to 880 nm with a 20 nm interval at a concentration of 0.125 mg/mL. The 808 nm NIR laser was purchased from Beijing Laserwave Optoelectronics. Both the DFT and the TDDFT calculations were conducted in the Gaussian 09 program on a CP trimer model using the B3LYP functional as well as the 6-31G(d,p) basis set.54 The alkyl side-chains are replaced with methyl chains to minimize the computational time. Synthesis of Conjugated Polymers. Synthesis of CP1. A solution of 4,7-diiodobenzo[c][1,2,5]thiadiazole (44.10 mg, 0.15 mmol), 4-(2hexyldecyl)-2,6-bis(trimethylstannyl)-4H-dithieno[3,2-b:2′,3′-d]pyrrole (109.4 mg, 0.15 mmol), Pd2(dba)3 (2.1 mg, 2.25 μmol), and P(o-tol)3 (2.7 mg, 9.00 μmol) in toluene (8 mL) was stirred at 90 °C under argon for 2 days. After the reaction was completed, the resultant polymer solution was cooled to room temperature and subsequently was precipitated in excess methanol. The precipitant was collected by centrifugation. The crude product was further washed with a large amount of methanol and acetone, respectively. Then the crude polymer was dissolved in chloroform and filtered with a short silica column. The 10131


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ACS Nano CCK-8 assay. For the in vitro PPT assay, U87 cells were cultured with CP3 NPs with different concentrations for 24 h. After removing free NPs, cells were exposed to NIR laser light (808 nm, 0.8 W/cm2) for 5 min. The photothermal cytotoxicity was assessed by the CCK-8 assay. In Vivo PA Imaging. The mice bearing subcutaneous U87 xenograft tumors were anesthetized with 2% isoflurane in oxygen and then located in prone position. Before and after intravenous injection of CP NPs/ ICG (0.5 mg/kg), the PA and ultrasound images of the tumor area were monitored via an AR-PAM system. In Vivo PTT. The balb/c nude mice bearing U87 tumors were separated into four groups with five animals per group as follows: (i) PBS group in which mice were only intravenously injected with PBS, (ii) NP group in which mice were only intravenously treated with NPs (2 mg/kg), (iii) laser group in which mice were only treated with NIR laser irradiation (808 nm, 0.8 W/cm2, 5 min), (iv) “NPs + laser” group in which mice were intravenously injected with CP3 NPs (2 mg/kg) and after 24 h of NPs injection were irradiated with an 808 nm laser (0.8 W/cm2, 5 min). When conducting PTT, the NIR laser beam spot was focused at the center of the mouse tumor. The tumor size and body weight were monitored every 3 days after PTT treatment. The tumor weight was calculated (tumor volume = length × (width)2/2). The measured volume value was normalized to evaluate the tumor volume. After PTT, tumors and major organs such as heart, liver, spleen, lung, and kidney were collected and sectioned for H&E staining. Toxicology Evaluation. Healthy Balb/c mice were separated into three groups with five mice in each group: (i) group 1 as a control, in which mice were untreated, (ii) group 2, in which mice were intravenously injected with 1 mg/kg of CP3 NPs, (iii) group 3, in which mice were intravenously injected with 5 mg/kg of CP3 NPs. At day 7 postinjection, the mouse blood (about 0.8 mL) was collected via the ocular vein and was characterized with a blood biochemistry assay as well as a complete blood panel test. H&E Staining. H&E staining was conducted following the BBC Biochemical protocol. First, prepared cryogenic slides (8 μm) were fixed with formalin (10%) for 30 min at room temperature. Later the slides were continuously cleaned using water for 5 min and further washed with alcohol with different concentrations from 100% to 95% and to 70% for 20 s in each concentration. The hematoxylin staining was carried out for 3 min and was washed for 1 min with water. Later the eosin staining was done in 1 min. Subsequently the slides were further washed with xylene and mounted by Canada balsam. A Nikon Eclipse 90i microscope was used to take images.

State Basic Research Development Program of China (973 Program, 2015CB755500), National University of Singapore (R279-000-482133), NRF Investigatorship (R279-000-444-281), Natural Science Foundation of China (81571745, 81401521, and 81327801), and Key Laboratory for Magnetic Resonance and Multimodality Imaging of Guangdong Province (2014B030301013). The authors declare no competing financial interest.

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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/acsnano.7b04685. Supplementary methods and results (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail (H. Zheng): [email protected]. *E-mail (B. Liu): [email protected]. ORCID

Bing Guo: 0000-0001-7981-0644 Lin Guo: 0000-0002-6070-2384 Bin Liu: 0000-0002-0956-2777 Author Contributions ⊥

B. Guo and Z. H. Sheng contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors are grateful for financial support from the Singapore NRF Competitive Research Program (R279-000-483-281), Major 10132


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