Highly Stable Conjugated Polymer Dots as Multifunctional Agents for

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Highly stable conjugated polymer dots as multifunctional agents for photoacoustic imaging-guided photothermal therapy Kaiwen Chang, Yubin Liu, Dehong Hu, Qiaofang Qi, Duyang Gao, Yating Wang, Dongliang Li, Xuanjun Zhang, Hairong Zheng, Zonghai Sheng, and Zhen Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00759 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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ACS Applied Materials & Interfaces

Highly

stable

conjugated

polymer

dots

as

multifunctional agents for photoacoustic imagingguided photothermal therapy Kaiwen Chang,1,3‡Yubin Liu, 1‡ Dehong Hu, 2 Qiaofang Qi, 1 ,3 Duyang Gao, 1 Yating Wang, 1 Dongliang Li, 1 Xuanjun Zhang, 1 Hairong Zheng, 2 Zonghai Sheng, 2* and Zhen Yuan1* 1 Faculty of Health Sciences, University of Macau, Macau SAR 999708, P. R. China 2 Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese of Academy of Sciences, Shenzhen 518055, China 3 Key Laboratory of Medical Molecular Probes, Department of Chemistry, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan 453003, China *Corresponding author: [email protected], [email protected] Keywords: conjugated polymer dots, multifunctional agents, theranostic nanoplatform, photoacoustic imaging, photothermal therapy

Abstract: Theranostic nanomedicines involved in photothermal therapy (PTT) have received constant attention as promising alternatives to traditional therapies in clinic. However, most photothermal agents are limited by their instability and low photothermal conversion efficiency. In this study, we report a new conjugated polymer dots (Pdots) as multifunctional agents for

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photoacoustic (PA) imaging-guided PTT. The novel BDT-IID Pdots are readily fabricated though nanoreprecipitation and can absorb strongly in 650-700 nm region. Furthermore, BDTIID Pdots possess the stable nanostructure and extremely low biotoxicity. In particular, its photothermal conversion efficiency can be up to 45%. More importantly, our in vivo results exhibit that the BDT-IID Pdots are able to offer concurrently enhanced PA contrast and sufficient photothermal effect. Consequently, the BDT-IID Pdots can be exploited as a unique theranostic nanoplatform for PA imaging-guided PTT of tumors, holding great promise for their clinical translational development.

Introduction

Cancer represents a serious public health concern and, for centuries, has been a leading cause of death around the globe.1-2 To improve the clinical outcome and survival rate of cancer patients, regular screening, surveillance programs and early intervention are widely recognized as the best methods used in cancer diagnosis and therapy. Conventional tumor therapies such as chemotherapy, radiotherapy, and surgery have proved successful, but also cause a variety of serious side effects to cancer patients during treatment.3-7 To improve treatment efficacy and reduce side effects, further efforts are now devoted to better identify different cancer therapeutic options that are effective, affordable, and acceptable to patients. Meanwhile, the development of non-invasive imaging technologies and image-guided tumor therapies is indispensable to improve the survival rate of cancer patients. Among these emerging techniques, photothermal therapy (PTT) has been developed into a powerful tool for noninvasive cancer theranostics owing to its high selectivity, generally low systemic toxicity, and negligible drug resistance.8-9 PTT strongly depends on the use of photothermal agents (PTAs) that provide an efficient


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conversion from light energy to heat in the thermal ablation of cancer cells and tumors.10-11 Interestingly, PTAs may also serve as contrast agents for the emerging diagnostic technique of photoacoustic (PA) tomography imaging. The latter has been reported as a method obtain functional information of biological tissues with higher resolution, higher contrast and deeper penetration depth compared to other optical imaging modalities.12-13 To date, various inorganic nanomaterials have been developed and applied to improve the PA imaging (PAI) contrast or enhance the efficacy of PTT, including noble metal nanomaterials (e.g. Au and Pd),14-17 transition metal dichalcogenides (e.g. MoS2, WS2 nanosheets, and CuS nanoparticles),18-20 and carbon nanomaterials (e.g. graphene and carbon nanotubes).21-22 However, one potential concern of these typically non-biodegradable inorganic nanomaterials is that they may linger in the body for a long period of time after injection, potentially causing long-term biotoxicity and therefore, significantly hindering potential in vivo applications. In contrast, compared to inorganic nanomaterials, organic PA/PTT agents represent more promising candidates for pre-clinical or clinical PAI/PTT due to the generally favorable biodegradability and biocompatibility characteristics. For example, indocyanine green (ICG), a FDA-approved contrast agent, has been extensively studied in the development of theranostic nanomedicines. ICG has been shown to eliminate effects of toxicity induced by exogenous probes.23,24 Although organic theranostic nanomaterials produced from near-infrared dyes can be produced via encapsulation strategies, they often show disadvantages of unstable nanostructures and insufficient photothermal conversion efficiencies.25 Therefore, the development of new organic nanomaterials as intrinsic theranostic nanomedicines with a robust nanostructure and high photothermal conversion efficiency for different PTT/PAI applications still remains a critical, albeit unmet, scientific goal.


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To date, conjugated polymers (CPs) as organic π-conjugated macromolecules have been explored as optically and electronically active materials for versatile optoelectronic devices and as nanoprobes for various biomedical applications.26-29 Specifically, as a novel category of photonic nanomaterials, π-conjugated polymer nanoparticles, also known as semiconducting polymer nanoparticles (SPNs)30,

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have attracted great interest as fluorescent probes for cell

tracking,32 tumor imaging,33,34 ultrafast hemodynamic imaging, and chemiluminescence imaging of drug-induced injury and neuroinflammation.35-37 This series of applications is most notably due to the excellent properties of high absorption, control dimensions and good biocompatibility.38-46 More importantly,π-conjugated polymer nanoparticles exhibit a high photothermal conversion efficiency, responsible for converting light energy to thermal energy and heat deposition in tumors, ultimately allowing for PTT.47-49 Meanwhile, π-conjugated polymer nanoparticles exhibit an extraordinary ability to convert light energy into acoustics. Therefore,

they may also serve as a flexible nanoplatform for in vivo PAI of tumors by

responsive to reactive oxygen species (ROS), enzyme, and pH.50-53 Furthermore, recent preliminary studies have demonstrated that polymer dots (Pdots) own utrla-stability.54-55 Herein, we report a novel Pdots-based PA/PTT agent in the 650~700 nm region for the PAIguided PTT of tumors. The produced BDT-IID Pdots exhibited several beneficial features for PA/PTT including: 1) robust nanostructure made from conjugated polymer; 2) high photothermal conversion efficiency; and 3) facile production by nanoreprecipitation method. The Pdots could be systematically characterized according to their PTT efficacy, optical absorption, size and structure, stability, cellular uptake and cytotoxicity. In addition, the cytotoxicity and therapeutic efficacy of the Pdots were evaluated in MCF-7 cells by using in vitro assays. The results indicated that the Pdots could efficiently damage cancer cells upon laser irradiation.


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Furthermore, the contrast enhancement for PAI by using Pdots was also successfully examined based on in vitro phantom experimental tests. More importantly, the in vivo PAI/PTT of tumors based on the developed Pdots was successfully implemented using a tumor-bearing mouse model.

The latter indicated that the tumors at different time points could be effectively

reconstructed with enhanced PA signals. Moreover, the tumor growth rates were also significantly inhibited or eradicated by PTT. Taken in concert, our results demonstrated that BDT-IID Pdots with a high photothermal conversion efficiency, strong optical absorption, and excellent stability may pave a new way for the enhanced PAI-guided PTT of tumors. Experimental Section Materials:4,8-Bis[5-(2-ethylhexyl)thiophen-2-yl]-2,6-bis(trimethylstannyl)benzo[1,2-b:4,5b ']dithiophene(BDT) and 6,6'-Dibromo-N,N'-(2-ethylhexyl)isoindigo (IID) were obtained from Derthon Optoelectronic Materials Science Technology Co LTD (Shenzhen, China).

The

functional copolymer poly (styrene-co-maleic anhydride) (PSMA, average MW ~1,700, styrene content 68%), Indocyanine green (ICG), Acridine orange (AO), Ethidium bromide (EB), Phosphate buffered saline (PBS) and Tetrahydrofuran (THF, anhydrous, 99.9%) were ordered from Sigma-Aldrich (Shanghai, China). (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)(MTT) were purchased from BioSharp (Hefei,China). Synthesis of BDT-IID conjugated polymer: A 4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]-2,6bis(trimethylstannyl)benzo[1,2-b:4,5-b']dithiophene (BDT) segment and 6,6'-dibromo-N,N'-(2ethylhexyl)isoindigo (IID) unit were used to synthesize the BDT-IID polymer as a typical donoracceptor (D-A)-type conjugated polymer via Stille coupling reaction. In a 25 mL flask, the mixture of monomer BDT (0.25 mmol, 226.14 mg) and monomer IID (0.25 mmol, 161.63 mg) was dissolved in 10 mL toluene. And then the mixture was degassed and recharged with nitrogen


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(N2) for five times before and after addition Pd(PPh3)4 (5 mg, 0.004 mmol). Under the protection of N2, the solution was stirred at 110 °C for 48 h. Further, the solution was allowed to cool to room temperature and was slowly added into methanol (150 mL) thereafter. Acetone (200 mL) was added into the solution including precipitates, followed by stirring overnight. Finally, the DA-type BDT-IID polymer was produced after solvent removal, and its number-average molecular weight was determined to be 141.56 kDa with a polydispersity index of 1.52. Preparation of BDT-IID Pdots: BDT-IID/PSMA Pdots were prepared using a nanoprecipitation method.56 To prepare BDT-IID Pdots, 0.8 mg of polymer BDT-IID and 0.2 mg of PSMA were mixed in tetrahydrofunan (THF, 1 mL). Then, the solution was added to 5.0 mL distilled water under ultrasonication. After THF removal via a stream of N2, the Pdots solution was obtained by filtration through a 0.22 micrometer filter to remove larger aggregates. Characterization of BDT-IID Pdots: TEM images of all samples were acquired on a standard transmission electron microscope (TEM, Hitachi H-600, Japan). Dynamic light scattering and Zeta potential measurements were performed on the Zetasizer NanoZS (Malvern, UK). All absorption spectra were measured using an UV−Vis 1700 spectrophotometer. 1H NMR spectra were recorded on a 500 MHz Bruker Avance instrument, using CDCl3 as solvent and tetramethylsilane (TMS) as internal standard (δ = 0.00 ppm). Number-average (Mn) and weightaverage (Mw) molecular weights of polymers were quantified by using a Waters Gel Permeation Chromatography (GPC) 2410 in trichloromethane (CHCl3) based on a calibration curve of polystyrene standards. Photothermal performance of BDT-IID Pdots:The photothermal performance for each of the dispersions containing BDT-IID Pdots at different concentrations (0, 50, 100, 150, 200, 250


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µg/mL) was individually measured upon 660 nm laser irradiation (200 mW/cm2) for a period of 600 s. For comparison, DI water was used as a control. In addition, the photothermal conversion efficiency of Pdots (100 µg/mL) was also studied systematically at different laser power (50, 100, 150, 200 mW/cm2) for a period of 600 s. During these measurements, temperature data was acquired every 10 s. The photothermal conversion efficiency of BDT-IID Pdots was generated by measuring the temperature change of Pdots aqueous dispersion as a function of time under the continuous irradiation of 660 nm laser (200 mW/cm2) for 600 s (t) till the temperature of solution reached a steady-state case. The photothermal conversion efficienty (η) was calculated by Eq. (1), η= [hA(TMax – TSurr) – QDis]/[I(1 – 10-Aλ)]

(1)

in which h is the heat transfer coefficient, A the surface area of the container, TMax the maximum steady-state temperature (53.2 °C), TSurr the ambient temperature of the environment (22.5 °C), QDis the heat dissipation from the light absorbed by the solvent and the quartz sample cell, I the incident laser power (200 mW/cm2), and Aλ the absorbance of the sample at 660 nm (0.446). The value of hA is computed by Eq. (2), hA= mDcD/τs

(2)

in which τs is the time constant for the heat transfer in the system, which was accessed based on the measurements in Figure 2d (τs = 145 s); and mD and cD, is the mass (0.5 g) and heat capacity (4.2 J/g) of the DI water used to disperse the Pdots, respectively. As a result, the hA was determined to be 14.48 mW °C-1.


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In addition, the heat dissipation from the light absorbed by the water and the quartz sample cells, QDis is measured independently to be 213.5 mW using a dispersible plastic cuvette cell containing distilled water. Consequently, the calculated photothermal conversion efficiency of the BDT-IID Pdots was 45%. In vitro cytotoxicity by MTT assay: The in vitro cytotoxicity of the Pdots was inspected by using MTT assay prior to the in vivo experiments. MCF-7 cells were seeded in DMEM supplemented with 10 % FBS (Gibco) and 1% penicillin/streptomycin (Gibco) for overnight. And then the original medium was totally removed from each well and subsequently DMEM (100 µL) containing specified concentrations of the Pdots was added to the designated wells. After incubation in the dark at 37 °C for 12 h, the cells incubated with the Pdots were irradiated with or without 660 nm laser (200 mW/cm2) for 10 min. The final concentration of the Pdots on each well plate ranged from 12.5 to 200 µg/mL. In addition, plates of MCF-7 cells were irradiated by using 660 nm laser with different power densities between 50 and 200 mW/cm2 for 10 min, which was to demonstrate that the laser irradiation itself had no cytotoxicity. All the plates were then incubated at 37 °C in the dark for 24 h. Further, we removed the old medium and added DMEM (100 µL without FBS) containing 10% MTT stock solution (5 mg/mL in sterile PBS) to each well. After 4h, the medium was removed completely, followed by adding dimethyl sulfoxide (DMSO, 100 µL) to each well. The cell viabilities were measured by using a BioTek Powerwave XS microplate reader (read the absorbance at 490 nm). The cells incublated with DMEM without any treatment represented 100% cell survival. AO/EB tests: The MCF-7 cells with the same cell density were cultured in four 35 mm dishes. After the overnight placement, the cells were respectively treated with PBS, only laser, only incubation with BDT-IID Pdots, and incubation with Pdots with laser irradiation (laser


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applied: 660 nm, 200 mW/cm2, 20 min). And then the cells were further incubated at 37 °C for 24 h. After staining with a mixture of AO/EB for 10 min and washing with PBS three times, the images of the four samples were generated by using a fluorescence microscope. In vivo PAI: The animal and phantom tests were performed by using the home-made multispectral PAI system in collaboration with the University of Macau. For this system, a pulsed light from an Nd:YAG laser (wavelength range from 680 to 1064 nm; pulse duration: 510 ns; frequency rate: 20 Hz; Surelite I-10, Continuum) was adopted as a laser source to illuminate the phantom or the animals through an optical subsystem. To record PA signals, a 1M transducer was circularly rotated by a rotary stage at 360 positions (1MHz central frequency; bandwidth range from 0.65~1.18MHz; V303-SU, Olympus-NDT). The complex wave field signal was first amplified by a Pulser/Receiver (5073R, Olympus) and subsequently converted into digital. Finally, the images were reconstructed by the delay-and-sum beam forming algorithm. For the phantom experimental tests, the target with different concentrations of BDT-IID Pdots was placed into the solid phantom, respectively. For the phantom materials, the agar powder (1-2%) solution was used to solidify the Intralipid as scatterer and India ink as absorber. Finally, the object-bearing solid phantom was immersed in water to measure the photoacoustic properties of Pdots. For all animal experiments, all protocols were approved by the Animal Management and Ethics Committee of the University of Macau. MCF-7 cells were cultured and subcutaneous animal tumor models were developed by subcutaneous injection of MCF-7 cells onto the back of the mice. All in vivo experiments were performed when the tumors reached a size of about


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80~140 mm3. The MCF-7 tumor-bearing mice were intravenously injected with Pdots at a dose of 1.5 mg/kg (body weight). PAI was performed at different time points (0, 1, 2, 4, 6, 8, 12, and 24 h) with a wavelength of 690 nm and using a water system at 37.5 °C. In vivo antitumor activity and biosafety: For all in vivo animal experiments, T-cell-deficient male nude (nu/nu) mice (5-6 weeks of age) as supplied by the Shanghai Slac Laboratory Animal Co. Ltd (Shanghai, China) were used with protocols approved by the Animal Management and Ethics Committee of the University of Macau. 5×106 MCF-7 cells were subcutaneously injected into the right legs of each mouse two weeks before commencing the treatment. Before PAI and PTT treatment, the tumors were allowed to reach a mean size of 80~140 mm3. The mice with tumors were equally divided into four groups (n = 5).

Subsequently, the mice received

intratumor injections of different formulations for different treatment strategies: (1) PBS; (2) Pdots; (3) Laser; and (4) Pdots + Laser. (Laser irradiation: 660 nm, 200 mW/cm2 for 10 min; concentration of various formulations: 100 µg/mL, 0.3 mL). The final injection dose was 1.5 mg/kg per mouse. Tumor volumes and mouse body weights were recorded every other day during treatment. After 21 days, the mice were sacrificed for further analysis. Tumor tissues and major organs were dissected for H&E staining thereafter. Results and Discussion The D-A-type conjugated polymer BDT-IID was synthesized through a Stille cross-coupling polymerization reaction. Figure 1a described the synthetic procedure of the conjugated polymer BDT-IID. The chemical structure was fully characterized by using 1H NMR and GPC analysis (Figure S1 and S2). The hydrophobic polymer BDT-IID and poly (styrene-co-maleic anhydride) (PSMA) were first dissolved into THF and then dispersed into water under ultrasonication,56 followed by the formation of Pdots after dialysis (Figure 1b). Dynamic light scattering (DLS)


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measurements were performed, and showed that the average hydrodynamic diameter of Pdots was around 20 nm (Figure 1c). The morphology of Pdots was characterized by using transmission electron microscopy (TEM), that showed that Pdots exhibited a uniform spherical morphology with an overall diameter of about 18 nm (Figure 1d). Importantly, we found that no aggregation and obvious changes in size occurred for the Pdots even after 30 days (Figure 1e), which demonstrated the high stability of Pdots in aqueous dispersions.54,

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Furthermore, the

surface potential of BDT-IID Pdots was approximately ~40 mV (Figure 1f), indicating that the BDT-IID Pdots were stable in aqueous solutions.58 In addition, there was no significant change in the surface potential of BDT-IID Pdots stored at room temperature for one months (Figure 1g). The UV–Vis absorption spectrum of Pdots showed a distinct absorption at the peak of 660 nm in the wavelength range of 650–900 nm (Figure 1h), a feature that proves to be highly relevant in light-triggered cancer therapy.


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Figure 1. a) Synthetic route of conjugated polymer BDT-IID. (i) Pd (PPh3)4 and toluene, 110 °C. b) Preparation of BDT-IID Pdots for PAI-guided PTT. c) Dynamic light scattering (DLS) measurement of the BDT-IID Pdots in water. d) TEM images of the as-prepared BDT-IID Pdots. e) Hydrodynamic diameters of BDT-IID Pdots as a function of storage at room temperature. f) Zeta potential of BDT-IID Pdots in water. g) Zeta potentials of BDT-IID Pdots as a function of storage at room temperature. h) Absorption spectra of BDT-IID Pdots dispersed in water.


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In order to examine the photothermal properties, the photothermal conversion efficiency of Pdots (100 µg/mL) was measured in aqueous solution under 660 nm laser irradiation. Figure 2a highlights a concentration-dependent temperature increase of the BDT-IID Pdots. This feature may be particularly important for PTT applications, in which BDT-IID Pdots show a temperature elevation when the concentration increased from 25 to 300 µg/mL. In addition, Figure 2b shows that the temperature of the solution increased upon enhancing the laser power. In particular, when the laser power reaches 200 mW/cm2, the temperature of the solution rapidly approached 53.2 °C, a feature that may effectively ablate tumor cells. However, this feature could not be observed in the case of the control group. Here, the temperature variation was negligible, as compared to the significant heat generation exhibited by the BDT-IID Pdots using the same irradiation dose. To measure the photothermal conversion efficiency of Pdots, we further monitored the temperature change of the BDT-IID Pdots dispersion under the continuous irradiation using a laser and an irradiation wavelength of 660 nm (200 mW/cm2). The laser was shut off when the temperature approached a stable level after irradiation for 600 s (Figure 2c). At a concentration of 100 µg/mL BDT-IID Pdots, the solution temperature was rapidly increased to 53.2 °C after laser irradiation for 600 s. The thermal images showed the photothermal effect and confirmed the same conclusion (Figure S3). Figure 2d plots the relationship between the cooling time versus negative natural logarithm of the temperature driving force generated during the cooling stage. The results from inspection of Figure 2d showed that the time constant for the heat transfer of the system was τs = 145 s. According to the generated results in Figure 2d, the photothermal conversion efficiency of the BDT-IID Pdots was calculated to be ~45%. More details are provided in the experimental section. As a comparison, photothermal conversion efficiency of


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the commercial Au nanorods with absorption peak at 650 nm was also measured under the same condition (Figure S4). It was calculated to be about 22%, which was similar with the previously reported.59,60 The photothermal conversion efficiency of other reported nanomaterials were listed in Table 1. It indicated that the as-prepared BDT-IID Pdots can be a good candidate for photothermal therapy. Furthermore, we found that even after seven irradiation cycles, the BDT-IID Pdots still featured the ability to increase the temperature to the same level (Figure 2e), suggesting excellent photostability characteristics of the BDT-IID Pdots, potentially enabling repeated PTT treatments. The photothermal stability was further compared to that of ICG, an FDA- approved commercially available dye used as an intravenously administered contrast agent with high optical absorption at wavelengths between 600-800 nm. As displayed in Figure 2f, no noticeable degradation in UV-Vis absorption of the BDT-IID Pdots could be observed, whereas the absorption of ICG was significantly reduced after 660 nm laser irradiation for 600 s. The figure inserts also show no observable color change in the dispersion of BDT-IID Pdots, whereas obvious fading from light green to colorless in the ICG solution could be determined. Taken in concert, the high photothermal conversion efficiency in combination with excellent photothermal stability of the BDT-IID Pdots may render the material useful as a highly effective PTT agent.

Table 1. Comparison of photothermal conversion efficiency of different nanomaterials Nanomaterials TDI-based Pdots BT-BIBDF Pdots Au-Cu9S5 NPs Au−attapulgite NPs Gold nanorods Gd:CuS@BSA NPs Gold-on carbon NPs

Photothermal conversion efficiency 41% 34.70% 37% 25.60% 21% 32.30% 31.60%

λmax, abs (nm) 640 782 1064 535,670 522,802 850

Ref. 61 62 63 59 60 64 65


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Figure 2. Photothermal properties of the BDT-IID Pdots. Photothermal heating curves of the BDT-IID Pdots a) with different concentrations upon 200 mW/cm2 laser irradiation at a wavelength of 660 nm and b) with different laser power densities at 100 µg/mL. c) Photothermal effect of the BDT-IID Pdots dispersions under laser irradiation at a wavelength of 660 nm (200


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mW/cm2). Irradiation was terminated after 600 s. d) Time constant for heat transfer was determined to be τs = 145 s by applying the linear time data from the cooling period (after 600 s) versus negative natural logarithm of driving force temperature, obtained from the cooling stage of (c). e) Temperature variations of the Pdots under irradiation at a power density of 200 mW/cm2 for seven light on/off cycles (10 min of irradiation for each cycle). f) Change of absorbance intensity of BDT-IID Pdots and ICG after repeated laser irradiation (n = 7). The figure inserts show the changes of BDT-IID Pdots and ICG after repeated laser irradiation (n = 7). To assess the in vitro cytotoxicity of BDT-IID Pdots as a PTT/PA agent, the measurements for the relative cell viabilities using MCF-7 cancer cells through 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays were performed. As shown in Figure 3a, upon 660 nm laser irradiation the BDT-IID Pdots demonstrated a dose-dependent cytotoxicity against MCF-7. However, this observation could not be made without laser irradiation, in which the cell viabilities remained nearly 90% for both cell lines, even at a high BDT-IID Pdots concentration of 100 µg/mL. Interestingly, the BDT-IID Pdots show the ability to enhance cell proliferation to some extent at the low concentration. This finding was in accordance with a study reported previously.66 Therefore, the results indicate an excellent biocompatibility of the Pdots. Since the Pdots also exhibited a superior cell killing ability to the MCF-7 cell line, we selected this cell line to inoculate mice for the following in vivo PTT experiments. Figure 3a shows the efficacy of 660 nm laser irradiation on the MCF-7 cell line without Pdots. We discovered that the laser irradiation itself has no direct effect on cytotoxicity and the cell death rates were due to the combined effects of Pdots upon laser irradiation. To further evaluate the cytotoxicity of the BDT-IID Pdots as a PTT agent, the viability of MCF-7 cells was evaluated by staining with acridine orange (AO) and ethidium bromide (EB) to


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differentiate live (green) and dead (red) cells. As illustrated in Figure 3b, compared with the control group (PBS only), the cells exhibited negligible cytotoxicity when only irradiated with laser or only incubated with the BDT-IID Pdots. However, we also found that the cells incubated with the BDT-IID Pdots and subjected to laser irradiation were mostly killed. These findings were consistent with the above MTT results, suggesting that BDT-IID Pdots have a remarkable cell-killing ability when subjected to laser irradiation and may therefore potentially serve as therapeutic agents for highly effective PTT. Histology analysis was further carried out to assess the in vivo toxicity of the as-obtained BDTIID Pdots before the potential in vivo biological applications. The major organ slices including the heart, liver, spleen, kidney, and lung were collected from mice sacrificed 7 days after intravenous injection of the BDT-IID Pdots for the histological analysis. No distinct damage was found in Pdots group and PBS group in the H&E staining images (Figure S5), indicating the assynthesized Pdots possessed histocompatibility at the dose tested.

Figure 3. In vitro cytotoxicity study against a) MCF-7 cells of Pdots with or without 660 nm laser irradiation at a power density of 200 mW/cm2 for 10 min. b) Fluorescence images of AO (green, live cells) and EB (red, dead cells) containing MCF-7 cells with PBS, laser, Pdots, and Pdots with laser irradiation.


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We further examined the PA properties of BDT-IID Pdots as contrast agents for PAI. The homemade multispectral PAI system is shown in Figure S6. The PA spectrum of BDT-IID Pdots was close to its absorption, showing maximum intensities at 690 nm (Figure 4a). Figures 4b and 4c showed that the corresponding PA intensities increased upon increasing the concentration of BDT-IID Pdots. Furthermore, the PA signals exhibited a linear correlation with the concentration of BDT-IID Pdots between 10 and 200 µg/mL (Figure 4d). These findings provide evidence for the advantages of BDT-IID Pdots as intrinsic theranostic nanomedicines for PAI. As shown in Figures 4e and 4f, the PA images of the tumor site were recorded at different time points after the intravenous injection of BDT-IID Pdots. Compared to the PA image before the injection of Pdots injection (0 h), the PA images showed a significant signal enhancement in the tumor region from 1 to 24 h post injection. The improved contrast at the tumor site was due to the enhanced permeability and retention (EPR) effect 67, indicating that Pdots may reach the tumor site after intravenous injection. Further analysis showed that the PA signals were the highest and the PAI contrast was the best between 4 and 8 h compared to the signals obtained from other time points after Pdots injection. For example, after 24 h post-injection, the PA signal intensity was found to decrease to its initial level, suggesting that BDT-IID Pdots were easily metabolized in the mice. After accumulated in the tumor, the concentration of the BDT-IID Pdots in blood was decreased. The P-dots can dissociate from tumor and then enter the lymphatic circulation or be taken up by hepatocytes from tumor, which should be attributed to their small size.68-70 The recovered results further validated that BDT-IID Pdots may serve as PA contrast agents for enhanced tumor imaging in vivo.


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Figure 4. PA properties of the BDT-IID Pdots. a) PA spectra of BDT-IID Pdots. b) PA images and c) corresponding PA signal intensities of BDT-IID Pdots dispersions of different concentrations (690 nm laser). d) Linear relationship between PA signal and concentration of BDT-IID Pdots. e) In vivo PA imaging of tumor tissue before and after tail injection of BDT-IID Pdots under 690 nm laser irradiation at different time points (0, 1, 2, 4, 6, 8, 12, and 24 h). f) Normalized PA signals in tumors at different time points. Encouraged by the remarkable in vitro cell-killing ability of BDT-IID Pdots in PTT, we carried out animal tests to demonstrate the in vivo antitumor activities of the material. As illustrated in Figure S7, infrared images of the MCF-7 tumor-bearing mice intratumorally injected with PBS or the BDT-IID Pdots and subjected to 660 nm laser irradiation (200 mW/cm2) for 600 s were generated, respectively. The corresponding temperatures of the tumors injected with BDT-IID Pdots increased rapidly upon laser irradiation and reached maximum at ~58.8 °C within 600 s, while the temperature of the tumors with laser irradiation alone exhibited only a minor temperature increase.


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To take advantage of the in vivo photothermal effect of the BDT-IID Pdots at the tumor site, PTT of the tumors was performed using four groups of mice (five mice in each group). Two groups of mice received intratumoral injections of PBS, whereas the other two groups were injected with the BDT-IID Pdots. The four groups were: 1) PBS; (2) Pdots; (3) Laser; and (4) Pdots + Laser. The tumors were further treated with a 660 nm laser irradiation for the PBS + laser group and Pdots + laser group, respectively. The in vivo PTT efficacy of BDT-IID Pdots was evaluated by measuring the tumor growth rate and the body weights of the mice. As shown in Figures 5a and 5b, the tumor volumes and body weights of mice treated with the different treatment strategies were recorded every other day, respectively, during a treatment period of 21 days. We found that the Pdots + laser treatment exhibited the most prominent tumor suppression and a complete tumor reduction was achieved. However, the mice in the other three groups exhibit no or only negligible tumor growth inhibition. These results demonstrated the therapeutic efficacy of Pdots upon laser irradiation was significantly high. More importantly, the mice in the other three control groups exhibited an average life spans of 16–28 days (Figure 5c), which was determined to be much lower than the average life span of mice in the Pdots + laser group. This increased average life span in the Pdots + laser group provided further evidence for the excellent PTT efficacy of Pdots. Moreover, Figure 5d showed representative photographs of tumor-bearing mice with different treatment strategies. We discovered that the mice that received Pdots in combination with laser irradiation treatment demonstrated the most effective tumor inhibition compared to from the mice in the other control groups. Likewise, the tumor sizes of the mice that received BDT-IID Pdots with laser irradiation treatment were also much smaller than those in the other three groups. These findings further confirmed the excellent PTT efficacy of BDT-IID Pdots with laser treatment. In addition, to help unveil the therapeutic effect of BDT-IID Pdots by


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PTT, hematoxylin and eosin (H&E)-stained sections from the heart, liver, spleen, lung and kidney of the mice in these groups (Figure 5e) were examined at autopsy. We discovered that no appreciable signs of organ damages or inflammatory lesions could be observed under the microscope, suggesting that the Pdots is low toxicity to the major organs during the PTT treatment. Our findings further indicate that BDT-IID Pdots exhibit excellent theranostic capabilities for PTT of tumors and excellent biological safety as well.

Figure 5. In vivo PTT effect of the BDT-IID Pdots. a) Tumor volumes and b) body weights of mice treated with PBS, Pdots, Laser, and Pdots + Laser. c) Survival curves of various groups of


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tumor-bearing mice after different treatments. d) Photograph of tumors after excision from different groups of mice at the end of treatments. e) Representative H&E staining images of major organs collected from mice after photothermal treatment. Conclusions In brief, we successfully developed highly stable BDT-IID Pdots as a contrast agent for PAI-guided PTT. The produced BDT-IID Pdots can be readily prepared and exhibited the robust nanostructures. In addition, the Pdots featured excellent biocompatibility characteristics and a high photothermal conversion efficacy up to 45%. The intrinsic theranostic properties to perform PAI with high spatial resolution and to mediate photoinduced tumor ablation were extensively studied in vivo. Furthermore, the BDT-IID Pdots not only showed a remarkable cell killing ability but also achieved a complete tumor regression, suggesting their excellent PTT efficacy. Finally, the BDT-IID Pdots exhibited excellent biosafety as well, rendering them a promising candidate for future clinical applications in PAI-guided PTT of various tumor species. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org, including the characterization (1H NMR, GPC) of BDT-IID polymer, thermal infrared images of Pdots, photothermal effect of the gold nanorods, H&E-stained images of major organs, homemade PA imaging system, and thermal infrared images of tumor-bearing mice with different treatments. AUTHOR INFORMATION Corresponding Author


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*Corresponding author: Z. Yuan ([email protected]); Z.Sheng ([email protected]) Present Addresses Dr.Kaiwen Chang and Miss Qiaofang Qi have moved to Key laboratory of Medical Molecular Probes, Department of Chemistry, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan 453003, China. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources MYRG2015-00036-FHS from the University of Macau, Major State Basic Research Development Program of China (973 Program)(Grant No. 2015CB755500), MYRG2016-00110FHS grants from the University of Macau, FDCT 026/2014/A1 and FDCT025/2015/A1 grants from Macao government in Macau, Natural Science Foundation of China (Grant Nos. 81771906, 81571745) and Key Laboratory for Magnetic Resonance and Multimodality Imaging of Guangdong Province (Grant No. 2014B030301013) ACKNOWLEDGMENT We gratefully acknowledge the financial support from the MYRG2015-00036-FHS from the University of Macau, Major State Basic Research Development Program of China (973 Program)(Grant No. 2015CB755500), MYRG2016-00110-FHS grants from the University of Macau, FDCT 026/2014/A1 and FDCT025/2015/A1 grants from Macao government in Macau, Natural Science Foundation of China (Grant Nos. 81771906, 81571745) and Key Laboratory for


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BRIEFS Highly stable conjugated polymer dots which were promising for combined photoacoustic imaging (PAI) and photothermal therapy (PTT) of cancer were developed. TOC


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Highly Stable Conjugated Polymer Dots as Multifunctional Agents for

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