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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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b04685 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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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,1‡ Zonghai Sheng, 2‡ Dehong Hu, 2 Anran Li, 3 Shidang Xu, 1 Purnima Naresh Manghnani, 1 Chengbo Liu, 4 Lin Guo, 3 Hairong Zheng2* and Bin Liu1* 1

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, Singapore 117585, Singapore 2

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, P.R. China 3

Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology, Ministry of

Education, School of Chemistry, Beihang University, Beijing 100191, P. R. China 4

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, P.R. China

ACS Paragon Plus Environment

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Corresponding Author Prof. Hairong Zheng. Shenzhen Institutes of Advance Technology, Chinese Academy of Science, 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, P. R. China 518055. Email address: [email protected] Prof. Bin Liu. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585. Email address: [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. ‡B. Guo and Z. H. Sheng contributed equally.

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


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obtained for U87 glioma cell ablation under laser irradiation (808 nm, 0.8 W/cm2, 5 min). This study shows that CP NP based PA/PTT theranostic nanoplatform is promising for future personalized nanomedicine. KEYWORDS: conjugated polymer nanoparticles, molecular engineering, theranostic platform, tumor, photoacoustic imaging, photothermal therapy

Theranostic 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 complicated fabrication process with compromised reproducibility and concerns of unstable nanostructures for in vivo applications. Therefore, it is of great value to develop “all-in-one” theranostic nanoplatform based on a single material which intrinsically contains both diagnosis and therapy functions.6-10 Among

different

theranostic

modalities,

light-activatable

nanoplatforms

with

dual

photoacousctic imaging (PA) and photothermal therapy (PTT) functionalities exhibit advantages over conventional theranostic nanoplatforms (e.g., fluorescence-guided chemotherapy). PA agents naturally posse photothermal functions, and they can synergistically offer deep penetration, high spatial resolution, and effective therapeutic function with minimal sideeffects.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 non-radiative exciton decay pathway to efficiently


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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 longterm 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 advantages 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 of 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 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 fluorescence in NPs.33-35 Most of these polymers, however, do not show optimized light absorption coefficient at the most widely used PTT excitation wavelength of 808 nm.33-35 Recently, we have developed donoracceptor (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


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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 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 down 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. Results and Discussion Synthesis of Conjugated Polymers CP1-3 and the NPs 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 CP1 < CP2 < CP3. CP1-3 dissolve well in organic solvents like 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


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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

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 and (e) Representative DLS result of CP3 NPs. 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


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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 CP13 exhibit planar structures with small dihedral angles in both all-trans- and all-cis 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/inter-chain 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 among the backbones, while the LUMO wave functions are more localized on the electron-deficient 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).


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


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Figure 3. (a-c) HOMO and LUMO wave functions of CP1-3 trimer with the geometry optimized structures (B3LYP/6-31G (d,p)) Characterization of Photo-Physical Properties of CP NPs Subsequently, CP1-3 were transformed into waster-dispersible NPs through nanoprecipitation using 1,2-distearoyl-sn-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 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/intra-chain 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


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absorption, as compared to CP1-2, indicating that the stiff structure leads to more intensified inter/intra-chain 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 L g-1 cm-1),19 Bi2Se3 nanosheet (11.5 L g-1 cm-1 L g-1 cm1 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), demostrating the strong capabilities of the CP NPs in light-harvesting.

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


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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 (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 can not only 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 o

C, while the temperature of CP1-2 NPs moderately approached 56.0 and 61.2 oC, respectively

(Figures 4e-f). The photothermal effect of CP3 NPs was further studied at different NP concentrations (Figure 4e and S7a). The photothermal temperature of CP3 NP suspension increases with the NP concentrations, which approaches a plateau temperature of 77 oC 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, 1h of irradiation with 808 nm laser (0.8 W/cm2) does not cause any obvious change in their absorbance or morphology (Figures S7b and S8-9). The cyclic photothermal heating and cooling processes also revealed that the NPs can be reversibly photothermal heated for five cycles without any noticeable change (Figure S8c). Under the same


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conditions, ICG can only reach a moderate temperature of around 34 oC (Figure S5c).The poor photothermal performance of ICG is mainly attributed to its severe photo-bleaching, which is confirmed by the absorbance loss after 5 min of continuous NIR laser irradiation (Fig S5d). Encouraged by the good photo-physical 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 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 (Fig 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 times of laser pulse irradiation (Figure 5f), which is highly desirable for PA imaging.


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Figure 5. In vitro PA study. (a) PA spectra of CP NPs and ICG (0.125mg/mL); (b) PA intensity of CP NPs and ICG (0.125mg/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. 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 was 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 a 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


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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. 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 cell growth. Once the U87 cells were treated with only 0.1 µg/mL of CP3 NPs, nearly 70% of cells were killed under the PTT treatment condition. The IC50 value was estimated to be 0.88 µg/mL under laser irradiation (808 nm, 0.8W/cm2, 5 min), which is much lower than the reported PTT agents (Table1 and Table S1), indicating great potentials of CP3 NPs for in vivo cancer cell ablation.

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


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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 co-stained 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).

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

Concentration

808 nm laser

µg/mL Cu2-xSe NPs 20

10

0.8 W/cm2, 5 min

CuS-ferritin nanocages 49

10

0.8 W/cm2, 5 min

Nano graphene oxide 50

6600

15.3 W/cm2, 8 min

Porphyrin-graphene oxide 51

100

0.8 W/cm2, 4 min

ICG NPs 52

10

1.25 W/cm2, 5 min

Polypyrrole microspheres 53

5

6 W/cm2, 5 min

0.88

0.8 W/cm2, 5 min

CP3 NPs

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 cross-section were synchronously monitored with a home-made 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


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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, at 24 h post-injection, 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 post-injection 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 post-injection of ICG, the PA signal in tumor reached the maximal contrast with a 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.


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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 of post injection with CP3 NPs; (c) Statistical results of PA signal in the tumor region at different time post injection of CP3 NPs.

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 subcutaneous U87 glioma-bearing mice model (Figure 8 and Figure S11). According to the PA imaging results, at 24 h post CP3 NP injection,


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the NPs reached high accumulation and penetrated into deep tumor region. As a consequence, the mice were treated with localized laser irradiation on tumor at 24 h post injection. Upon laser irradiation, the tumor temperature of mice treated with CP3 NPs rapidly jumped to 52.7 oC within 5 min, while the control group exhibited only a slight temperature increase to 34.5 oC 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 could affect tumor growth (Figure 8e). These results confirm that the CP3 NPs are capable to mediate effective photothermal destruction on 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 growth with 0% of survival rate on day 21, suggesting that neither NPs injection alone nor laser treatment alone can inhibit tumor growth. In contrast, for CP3 NP-mediated PTT treatment group, the growth of U87 tumor was totally eliminated and no tumor recurrence was found with survival rate of even 100% on day 30. Moreover, during PTT treatment process, neither significant body weight loss nor obvious body weight difference were observed among all groups, indicating insignificant side-effects of the treatment process. The excellent biocompatibility and high tumor ablation efficiency via PTT in vivo are consistent with the results in vitro.


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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.

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 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


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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).

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


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Conclusion In summary, we synthesized and systematically studied the photo-physical 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 Materials. 4,7-Dibromobenzo[c][1,2,5]thiadiazole (BT) was provided by Puyang Huicheng and was purified via recrystallization in methanol.4-(2-Hexyldecyl)-2,6-bis(trimethylstannyl)-4Hdithieno[3,2-b:2',3'-d]pyrrole

(DTP)

and

3,6-bis(5-bromothiophen-2-yl)-2,5-bis(2-

octyldodecyl)pyrrolo[3,4-c]pyrrole-1,4 (2H,5H)-dione(DPP) were supplied by Solarmer, Beijing, China. 4,7-Dibromo[1,2,5]thiadiazolo[3,4-c]pyridine (NBT) and 4,8-dibromo-6-(2-ethylhexyl)[1,2,5]thiadiazolo[3,4-f]benzotriazole (TBZ) were obtained from Derthon, Shenzhen, China. 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPEPEG2000) 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 tetrahydrafuran were obtained from Fisher. All other chemicals were used as received.


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Characterization. NMR spectra were characterized with a Bruker 500 NMR spectrometer. In NMR spectra, the chemical shifts of the residual solvent peaks and TMS at δ = 0.00 ppm were used as reference. Gel permeation Chromatography (GPC) results were tested via Waters 996 photodiode detector and aphenogel GPC column. The running conditions are polystyrene standard in THF eluent at 1.0 mL/min at room temperature. UV-vis spectra were studied with Shimadzu Model UV-1700 spectrometer. PL spectra were tested using a Perkin-Elmer 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 home-made 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 the concentration of 0.125 mg/mL. The NIR laser of 808 nm was purchased from Beijing Laserwave Optoelectronics. Both the density functional theory (DFT) and the timedependent DFT (TDDFT) calculations were conducted in the Gaussian 09 program on CP trimer model using the B3LYP function 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, 0.15mmol), 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 oC under argon for two days. After the reaction was completed, the resultant polymer solution was cooled down to room temperature and subsequently was precipitated in excess methanol. The precipitant was collected by centrifugation. The crude product was further washed with large amount of methanol and acetone, respectively. Then the crude polymer was


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dissolved in chloroform and filtered with a short silica column. The polymer solution was carefully concentrated via rotary evaporator and was washed with excess methanol. The precipitant was dried in vacuum to yield CP1 as a dark solid (52 mg, yield 65%). 1H NMR (500 MHz, C2D2Cl4, 100 oC) 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[3,4-c]pyridine (44.24 mg, 0.15mmol), 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 oC under argon for two 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 oC) 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)-2,5-bis(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 oC under argon for two 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 oC) δ 8.87 (br, 2H), 7.48-7.04 (br, 4H), 4.11 (br, 4H), 2.08 (br, 2H), 1.451.32 (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.


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Preparation of CP NPs. For the synthesis of CP NPs, conjugated polymer (0.5 mg) and DSPEPEG2000 (1 mg) were completely dissolved in 1 mL of THF. The resultant solution was further sonicated for ten minutes in 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 seconds. After sonication, the mixture was stirred in fuming 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 for 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 NPs suspension at 0.0125 mg/mL were under NIR laser irradiation (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 duration 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 Medium (DMEM) media in a humidified environment with 5% CO2 at 37 oC. The DMEM was added with 10% of fetal bovine serum and 1% of penicillin streptomycin. Before starting experiments, the cells were precultured until confluence was achieved. All animal studies were conducted according to the animal use and


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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 tumor-bearing nudemice, U87 cells (3 × 106) in PBS (100 µL) were subcutaneously injected at 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 cytotoxicity assay, 5×104 of 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 standard CCK-8 assay. For 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 CCK8 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 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) NPs 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


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mg/kg), and after 24h of NPs injection were irradiated with 808 nm laser (0.8 W/cm2, 5 min). When conducting PTT, the NIR laser beam spot was focused at the center of 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 post injection, the mouse blood (about 0.8 mL) was collected via the ocular vein and was characterized with blood biochemistry assay and as well as 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.


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ACKNOWLEDGMENT The authors are grateful for financial support from the Singapore NRF Competitive Research Program (R279-000-483-281), Major State Basic Research Development Program of China (973 Program, 2015CB755500), National University of Singapore (R279-000-482-133), 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.

Supporting Information. Supplementary methods and results are available free of charge via the Internet at http://pubs.acs.org.

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Improved

Charge

Transport

and

Absorption

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