Near-Infrared (NIR)-Absorbing Conjugated Polymer Dots as Highly

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Near-Infrared Absorbing Conjugated Polymer Dots as HighlyEffective Photothermal Materials for In Vivo Cancer Therapy Shengliang Li, Xiaoyu Wang, Rong Hu, Hui Chen, Meng Li, Jianwu Wang, Yunxia Wang, Libing Liu, Fengting Lv, Xing-Jie Liang, and Shu Wang Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b03738 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 5, 2016

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Chemistry of Materials

Near-Infrared Absorbing Conjugated Polymer Dots as Highly-Effective Photothermal Materials for In Vivo Cancer Therapy Shengliang Li,a Xiaoyu Wang,a Rong Hu,a Hui Chen,a Meng Li,a Jianwu Wang,a Yunxia Wang,a Libing Liu,a Fengting Lv,a Xing-Jie Liang*b and Shu Wang*a a

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China. E-mail: [email protected]

b

Chinese Academy of Sciences Center for Excellence in Nanoscience, CAS Key Lab for

Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, P. R. China Email: [email protected]

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ABSTRACT: Photothermal therapy (PTT) holds great promise for non-invasive cancer treatment. To fulfill this goal, highly effective and low-risk photothermal agents have been intensively explored. Here we present a new PTT material based on conjugated polymer dots (Pdots) that exhibit strong near-infrared (NIR) absorption and high photostability. The Pdots result in a thermal response upon illumination with a NIR laser, leading to a high photothermal conversion efficiency of 65%. Thus, the photothermal ablation of cancer cells using the Pdots both in vitro and in vivo can be achieved, highlighting the potential of Pdots as a nanoplatform for clinical therapy. They also open up a new avenue to develop new photothermal therapeutic materials.

INTRODUCTION Conjugated polymers (CPs), characterized by electron delocalized backbones and rapid exciton diffusion, also along the backbone, have been widely used in optoelectronic devices, such as organic field-effect transistors,1-3 organic solar cells,4,5 and organic light-emitting diodes.6,7 Recently, owing to their excellent light-harvesting and light-amplifying properties, the application of CPs is further being expanded into biomedical fields, including optical and electronic biosensors,8-11 bio-imaging,12-15 and anticancer and antimicrobial therapies.16-19 In particular, conjugated polymer dots (Pdots) of small sizes have drawn extensive attention because of their intriguing optical characteristics and nontoxic features for living systems.20-23 There are enormous efforts to develop Pdots that have multicolor and near-infrared (NIR) emission as fluorescent probes for bioimaging and sensor design. Moreover, Pdots have begun to see development as a light-activated agent, and have been applied for anticancer and antimicrobial phototherapy.24-26 However, these light-activated activities mainly rely on visible light, and thus the penetration ability and phototoxicity of visible light prevents further biomedical applications.


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Through altering the donor-acceptor (D-A) construction units in Pdots, their absorption and fluorescence emission can be finely regulated, providing a strategy to address the above limitations.27,28 The extension of absorption and emission of Pdots to NIR (λ=700-1350 nm) will ensure their potential applications in biomedical area, such as NIR imaging and NIR-activated phototherapy.29,30 Moreover, taking advantage of non-invasive

and

spatiotemporal-controlling modes, the biodegradable Pdots with NIR-induced photoactivity exhibit great potential for in vivo cancer therapy. Herein,

four

photothermal

Pdots

are

developed

using

diketopyrrolopyrrole

(DPP)-containing CPs bridged with different thiophene units (see their chemical structures in Figure 1), which display strong NIR absorbance and high photothermal conversion efficiency for in vivo photothermal therapy of tumor. Particularly, the relationship of donor-acceptor backbone structures in Pdots with their optical and photothermal properties is studied. These Pdots exhibit efficient conversion ability from photon energy into heat, leading to a high photothermal conversion efficiency of 65%. A proof-of-concept application of Pdots as good photothermal materials is thus demonstrated for in vitro and in vivo tumor treatments.

RESULTS AND DISCUSSION Synthesis and Characterization of CPs 1-4. The electron-deficient feature of DPP provides a possibility to adjust its optical band gap into NIR region via copolymerization with electron-rich aromatic heterocycles.31-34 Based on this strategy, four D-A CPs containing DPP and various thiophene derivatives (monothiophene, thienothiophene, bithiophene, and benzodithiophene) were synthesized by Stille coupling reaction (Figure S1-4, Supporting Information). Their molecular structures are depicted in Figure 1b. Due to the strong electron-withdrawing ability of DPP, these CPs exhibit broad absorption spectra ranging from 600 to 900 nm in chloroform solution, as shown in Figure 1c. Advantageously, the energy band and optical property could be regulated 3 Environment ACS Paragon Plus


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effectively by changing thiophene donors, and the maximim absorption peaks of CPs 1-4 are 816, 810, 790, and 750 nm, respectively (Figure 1d and Figure S5, Supporting Information). To evaluate their NIR absorbing capability, the corresponding mass extinction coefficients at 808 nm were further measured, which is 72.9, 60.6, 55.6, and 33.0 Lmg−1 cm−1, respectively (Table 1). Meanwhile, all CPs showed almost no fluorescence emission, indicating that the non-radiative decay is the main pathway for excited state deactivation.

Preparation and Characterization of Pdots. For further PTT application, Pdots 1-4 were respectively prepared from the hydrophobic CPs 1-4 by nanoprecipitation method (Figure 1a). The morphologies of Pdots were determined by transmission electron microscopy (TEM) (Figure 1e), and the results showed that Pdots were uniform spherical shape with a diameter of about 30 nm. Dynamic light scattering (DLS) measurement presents a hydrodynamic diameter of 49 ± 3 nm with a polydispersity index (PDI) value of 0.19, indicating the monodisperse preference of Pdots in aqueous solution (Figure 1f). Besides, these Pdots did not show any aggregation behavior and maintained homogeneous size even storage at 4 °C for one-month, indicating their good stability (Figure 1g). Photophysical properties of Pdots were also investigated and the results showed that the extinction coefficient and fluorescence quantum yield of Pdots were similar to those of CPs in chloroform solution.


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Figure 1. a) Schematic illustration of preparation for Pdots. b) Molecular structures of CPs 1-4. c) UV-Vis-NIR absorption spectra of CPs1-4 in chloroform. d) Band diagram representing the HOMO and LUMO levels of CPs 1-4 determined by cyclic voltammetry. e) Representative TEM images of Pdot-1. f) Size distribution histogram of Pdot-1 by DLS measurement. g) Photographic images of Pdots 1-4 in aqueous solutions (25 µg/mL) after one-month storage at 4 °C.



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Table 1. Molecular weight and photophysical properties of CPs 1-4. Polymer

Mn (Kg/mol)

PDI

λMax, abs (nm)

−1

ε808 (cm g−1 L)

QY (%)

CP1

64.1

2.3

816

72.9

0.1

CP2

37.3

2.5

810

55.6

0.2

CP3

52.8

1.9

790

60.6

0.2

CP4

45.1

3.6

750

33.0

0.1

Photothermal Performance of Pdots In consideration of the strong NIR absorption of these Pdots, we further studied their photothermal performances. Sharp temperature increases were observed upon irradiation with an 808 nm laser at 0.5 W/cm2 for 5 min, indicating that the Pdots possess good photothermal response (Figure S6, Supporting Information). Moreover, Pdot-1 shows the best photothermal performance among these Pdots, possibly due to its higher mass extinction coefficient at 808 nm (Table S1, Supporting Information). Thus, the photothermal conversion capacity of Pdot-1 was chosen and further investigated in following experiments (Figure 2a and b). The temperature of the Pdot-1 aqueous solution increases with irradiation time extension and concentration increase. At low concentration (50 µg/mL) of Pdot-1, the temperature increased up to 61.4 °C after 5 min irradiation, while that of the pure water slightly changed, confirming the fast and high-efficient photothermal conversion capacity of Pdots (Figure 2b). The photothermal response of Pdot-1 under various laser densities was also investigated and showed laser density-dependent profile (Figure S7, Supporting Information). The photothermal conversion efficiency of Pdot-1 was calculated to be approximately 65%


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(Figure 2c and Figure S8, Supporting Information) using previlously reported method,35 which is higher than those of Au NRs (21%),35 CuS nanocrystals (25.7%),36 black phosphorus (28.4%),37 and melanin nanospheres (40%)38. The photothermal conversion efficiencies of Pdots 2-4 were also calculated to be approximately 55%, 42%, and 34%, respectively. Compared with other reported conjugated polymers or their nanoparticles, Pdots in this work show higher photothermal conversion efficiency because of the reduced energy loss from excitation state.39-41 As we known, the photothermal stability is an important parameter in photothermal therapy. In order to further investigate the photothermal stability of Pdots, the samples were irradiated by the 808 nm laser for 5 min and then turned off for cooling down to room temperature, and the ON/OFF cycle was repeated five times. ICG, the FDA-approved NIR dye widely used for clinical phototherapy, was chosen as control. As shown in Figure 2d, after five ON/OFF cycles of irradiation at 0.5 W/cm2, Pdots still maintain high photothermal effect while that of ICG loses mostly under the same condition. It is noted that the NIR absorbing ability of Pdots does not change in this process, while that of ICG disappears completely after five cycles (Figure S9, Supporting Information). Importantly, after five ON/OFF cycles of irradiation, the size of Pdots maintain their narrow size distribution, with an average size of 48.7 nm (Figure S10, Supporting Information). The above results confirm the high and stable photothermal performance of Pdots, and highlight their good potential as photothermal materials for PTT treatment.



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Figure 2. Infrared thermographs a) and temperature elevation b) of water and Pdot-1 aqueous solutions with different concentrations as a function of irradiation time. c) Photothermal response of Pdot-1 aqueous solution (25 µg/mL) under irradiation for 5 min with an NIR laser (808 nm, 0.5 W/cm2) and then the laser shut off. d) Temperature profiles of Pdot-1 aqueous solution for five ON/OFF cycles.

In vitro Photothermal Ablation of Cancer Cells The potential of Pdots as PTT materials for in vitro ablation of cancer cells was evaluated due to their favorable photothermal effect as demonstrated above. As a proof-of-concept experiment, 4T1 cells were chosen as cancer cells model. 4T1 cells were incubated with Pdot-1 and then irradiated by 808 nm laser at 0.5 W/cm2 for 5 min, and then the live/dead cells were determined after treatment by calcein AM and propidium iodide (PI) (Figure 3a-d). After irradiation, the distinct boundary between green (live cells) and red (dead cells) was detected under confocal laser scanning microscopy (CLSM), and the cells within the laser region displayed red fluorescence, indicating that the cells were killed completely 8 Environment ACS Paragon Plus

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after PTT treatment. The standard MTT was employed to confirm and quantify the photothermal ablation effect. As shown in Figure 3e, after incubation with various concentrations of Pdot-1 for 24 h in dark, no obvious cytotoxicity and proliferation inhibition of 4T1 cells were observed even the concentrations of Pdots up to 100 µg/mL. However, the 4T1 cells were efficiently killed by Pdots under irradiation, and more than 90% cells were ablated even under the concentration as low as 25 µg/mL (Figure 3f). These results indicate that Pdots have perspectives as highly efficient and low toxic PTT materials for in vivo cancer treatment.

Figure 3. a) Thermal images of the 4T1 cell culture dish after incubation with Pdot-1 and laser irradiation (0.5 W/cm2) for 5 min. b-d) Fluorescence images of cells stained with calcein AM (live cells, green fluorescence) and PI (dead cells, red fluorescence). e) Cell viability of 4T1 cells after incubation with various concentrations of Pdot-1 for 24 h. f) Relative viabilities of 4T1 cells after treatment with Pdot-1 under 808 nm laser at 0.5 W/cm2 for 5 min.

In light of the good NIR absorption and photothermal conversion of Pdots, we then carried out in vivo PTT treatment using Pdots in the 4T1 tumor-xenograft model. The mice bearing tumors were intratumorally injected with Pdots (0.5 mg/mL, 40 µL) and then 9 Environment ACS Paragon Plus


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irradiated under 808 nm laser. The temperature change in tumors under laser irradiation was monitored by infrared thermal imager. As shown in Figure 4a and 4b, upon irradiation at the power densities of 0.25 and 0.5 W/cm2, the tumor temperature raise to 53-68 °C within 5 min, which is high enough to kill tumor in vivo. As a control, the tumor temperature is slightly altered under the same laser condition when the mice were treated by saline injection.

In Vivo Photothermal Therapy We then studied the in vivo photothermal therapeutic efficacy of Pdots. After the tumor size achieved approximately 60 mm3, the 4T1 tumor-bearing mice (6 mice per group) were divided into four groups. In the treatment group, the mice were intratumorally injected with Pdot-1 (0.5 mg/mL, 40 µL) and followed with 5 min irradiation by the 808 nm laser at a power density of 0.5 W/cm2. Other control groups of mice included untreated mice, mice administered with Pdots without laser irradiation, and mice irradiated with laser only. The tumor sizes were measured by an electronic digital caliper on other day.

As shown in

Figure 4c and 4e, after Pdots injection and laser irradiation, the tumors were effectively ablated and only black scars were left at tumor lesions. Notably, no recurrent was found in Pdots-induced PTT treatment group even for further 14 days feeding. Nevertheless, other three control groups have homologous tumor growth ratio, which indicates that neither Pdots nor laser irradiation could hinder the tumor growth. More importantly, all mice in treated group owned life spans over 30 days while mice in control groups just survived 14-25 days in average (Figure 4d). These findings confirm that Pdots possess powerful potential for in vivo PTT cancer treatment.


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Figure 4. a) Thermal images of tumor-bearing mice exposed to the 808 nm laser irradiation for 5 min after the injection of saline and Pdot-1. b) Time-dependent photothermal heating curves of tumor upon laser irradiation as a function of irradiation time. c) Tumor growth curves in different groups of tumor-bearing mice after various treatments. Relative tumor volumes were normalized to their initial sizes. Error bars represent the standard deviation of 6 mice per group. Asterisk indicates P

Near-Infrared (NIR)-Absorbing Conjugated Polymer Dots as Highly

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