Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy

Accepted Manuscript Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy Haobin Chen, Jian Zhang, Kaiwen Chang, Xiaoju Men, Xiaofeng Fang, Libo Zhou, Dongliang Li, Duyang Gao, Shengyan Yin, Xuanjun Zhang, Zhen Yuan, Changfeng Wu PII:

S0142-9612(17)30509-4

DOI:

10.1016/j.biomaterials.2017.08.007

Reference:

JBMT 18208

To appear in:

Biomaterials

Received Date: 8 May 2017 Revised Date:

6 August 2017

Accepted Date: 7 August 2017

Please cite this article as: Chen H, Zhang J, Chang K, Men X, Fang X, Zhou L, Li D, Gao D, Yin S, Zhang X, Yuan Z, Wu C, Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.08.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Article type: Original Research

Highly Absorbing Multispectral Near-Infrared Polymer Nanoparticles from One Conjugated Backbone for Photoacoustic Imaging and

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Photothermal Therapy

Haobin Chen a,b, Jian Zhang c, Kaiwen Chang b,c, Xiaoju Men a,b, Xiaofeng Fang b, Libo Zhou a,

a

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Changfeng Wu b, *

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Dongliang Li c, Duyang Gao c, Shengyan Yin a, Xuanjun Zhang c, Zhen Yuan c, and

State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun, Jilin 130012, China

b

Department of Biomedical Engineering, Southern University of Science and Technology,

Corresponding author:

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*

Faculty of Health Science, University of Macau, Taipa 999078, Macau

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c

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Shenzhen, Guangdong 518055, China

Changfeng Wu

E-mail adress:

[email protected]

Fax number:

(86) 0755-88015137

Postal address:

Department of Biomedical Engineering Southern University of Science and Technology Shenzhen, Guangdong 518055, China

1

ACCEPTED MANUSCRIPT ABSTRACT Semiconducting polymers with specific absorption are useful in various applications, including organic optoelectronics, optical imaging, and nanomedicine. However, the optical

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absorption of a semiconducting polymer with a determined structure is hardly tunable when compared with that of inorganic semiconductors. In this work, we show that the optical absorption of polymer nanoparticles from one conjugated backbone can be effectively tuned through judicious design of the particle morphology and the persistence length of polymers.

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Highly absorbing near-infrared (NIR) polymers based on diketopyrrolopyrrole-dithiophene

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(DPP-DT) are synthesized to have different molecular weights (MWs). The DPP-DT polymer with a large molecular weight and high persistence length exhibited remarkably high optical absorption with a peak mass extinction coefficient of 81.7 L g-1 cm-1, which is one of the highest value among various photothermal agents reported to date. Particularly, the polymer nanoparticles with different sizes exhibit broadly tunable NIR absorption peaks from 630 to

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811 nm. The PEGylated small polymer dots (Pdots) show good NIR light-harvesting efficiency and high non-radiative decay rates, resulting in a relatively high photothermal

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conversion efficiency in excess of 50%. Thus, this Pdot-based platform can serve as

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promising photothermal agents and photoacoustic probes for cancer theranostics.

KEYWORDS

photothermal therapy; photoacoustic imaging; near-infrared absorption; polymer dots; contrast agents.

2

ACCEPTED MANUSCRIPT 1. Introduction Organic semiconductor materials have received considerable attention in recent years owing to their fascinating physical properties and extensive applications [1, 2].

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Semiconducting polymers are macromolecules characterized by a system of delocalized πelectrons by hopping, tunneling, and related mechanisms, which can result in intriguing and tunable optical and electrical properties [3]. Owing to these outstanding characteristics, semiconducting polymers have been widely used in photonics, sensing, solar energy

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conversion, and biomedicine [4-6]. There is also a great deal of interest in the development of

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nanoparticle systems given their broad potential applications in biological medicine [7]. In particular, semiconducting polymer dots (Pdots) represent an emerging nanotechnology in the energy and health fields [8-11]. Pdots exhibit high brightness and rapid emission rates, show excellent photostability, and are nontoxic, with demonstrated utility in a wide range of applications such as cellular labeling [12, 13], in vivo imaging [14, 15], photoacoustic

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imaging [16, 17], single-particle tracking [18], and biosensing [19, 20]. Semiconducting polymer-based multifunctional nanoparticles have also been demonstrated to have theranostic

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applications because of their flexible matrix for accommodating drugs, photosensitizers, and other imaging contrast agents [21-23].

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Photothermal therapy (PTT) is an attractive therapeutic methodology that employs the conversion of absorbed near-infrared (NIR) light into thermal energy to ablate cancerous cells. PTT has received tremendous attention in the field of cancer therapy because of its specific selectivity to cancer-targeting sites and minimal invasion to the surrounding normal tissues. Thus, it would be highly desirable to shift the wavelength of irradiation light to the “therapeutic window” (600–900 nm) to achieve the maximum penetration depth for solid tumors, while minimizing the scattering and absorption of photons by the tissue in order to reduce damage to surrounding healthy tissues. Currently, many light-absorbing inorganic and 3

ACCEPTED MANUSCRIPT organic photothermal agents are being extensively explored as efficient photothermal materials for in vivo cancer therapy, such as gold nanomaterials [24-26], carbonous nanostructures [27, 28], transition-metal dichalcogenides [29, 30], copper sulfide nanoparticles [31], magnetic nanoparticles [32], and organic dyes [33] and polymers [34].

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However, most of these thermotherapy agents are not utilized in clinical practice due to their potential long-term toxicity and poor drug metabolism and pharmacokinetic (DMPK) properties. Therefore, the development of therapeutic agents that can serve as imaging agents

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to monitor the biodistribution and DMPK of these photothermal probes is essential. Recently, researchers designed and synthesized a class of biocompatible conjugated polymer

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nanoparticles as highly effective photothermal agents for the PTT ablation of cancer in vivo [35, 36]. Pu et al. proposed an intraparticle molecular orbital engineering approach to enhance both the photoacoustic brightness and PTT efficiency of semiconducting polymer nanoparticles for amplified theranostics [37]. In another recent report, Liu et al. developed

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nanoscale metal-organic particles with rapid clearance for the imaging-guided PTT of cancer [38]. These are encouraging achievements in cancer therapeutics and have stimulated the extensive development of NIR photothermal techniques.

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Photoacoustic imaging (PAI), as an emerging noninvasive biomedical imaging modality based on optical excitation and ultrasound detection [39], has attracted considerable interest

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primarily because of its superiority to traditional optical imaging technologies, e.g., low acoustic scattering, nonionizing radiation, high depth-to-resolution ratio, high sensitivity, multiscale capacity from organelles to organs, and inherent background-free and speckle-free features [40-43]. In particular, PAI has the capability to provide structural, functional, and molecular information of biological tissues and has enabled the clear visualization of deep tumors in preclinical and clinical studies [44-47]. Because both photoacoustic and photothermal approaches involve thermoplastics, it may be useful to integrate PAI and PTT using the same probes, thereby permitting imaging-guided therapy. Therefore, an ideal 4

ACCEPTED MANUSCRIPT photoacoustic and photothermal agent should have a large absorption coefficient, good biocompatibility, good photostability, and well-controlled surface functionalization, which are important characteristics for clinical practice in cancer therapeutics. In this work, we designed and synthesized the highly absorbing NIR polymer (DPP-DT)

with

different

molecular

weights.

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diketopyrrolopyrrole-dithiophene

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nanoparticles of different sizes were characterized for their optical absorption, NIR lightharvesting efficiency,

and photothermal conversion efficiency. Intriguingly, these

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nanoparticles from one conjugated backbone showed intense tunable absorption from 630 nm to 811 nm. The polymer with high persistence length exhibited remarkably high optical

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absorption with a peak mass extinction coefficient of 81.7 L g-1 cm-1, which was much higher than most of photothermal agents. In vitro cellular assays and in vivo animal experiment indicate these Pdots are promising PTT and PAI contrast agents for photoacoustic imaging

2. Results and discussion

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and photothermal cancer treatment.

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2.1. Design and synthesis of highly absorbing NIR semiconducting polymers The specific optical absorption of semiconducting polymers is critical for their optoelectronic

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and biomedical applications. Because of the planar structures and strong electron deficiency, the value of diketopyrrolopyrrole derivatives has been increasingly recognized in the development of organic optoelectronic devices. Nelson et al demonstrated that semiconducting polymers with higher MWs (120 kDa) led to substantially enhanced mass extinction coefficients that were 1.4-fold larger than those of fractions with lower MWs (20 kDa) [48]. Unfortunately, the high molecular weights of semiconducting polymers always cause poor solubility in tetrahydrofuran, preventing subsequent nanoprecipitation. In this context, we designed and synthesized a series of long side-chain (tetradecyl-decyl) polymers, 5

ACCEPTED MANUSCRIPT poly(diketopyrrolopyrrole-alt-dithiophene) (DPP-DT), by Stille polymerization according to the synthetic route shown in Fig. 1a. DPP-DT polymers were characterized by nuclear magnetic resonance (NMR) spectra (Fig. S1, Supporting Information), Fourier transform infrared (FTIR) spectra (Fig. S2, Supporting Information), and gel permeation

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chromatography (GPC) (Fig. S3–5, Supporting Information). As the MW affects the absorption coefficient, we synthesized three DPP-DT polymers with different MWs (DPPDT-H, DPP-DT-M, and DPP-DT-L, respectively) by varying the polymerization time. GPC

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measurements revealed that DPP-DT-H had a number average MW (Mn) of 153 kDa with a polydispersity index (PDI) of 1.73, DPP-DT-M had an Mn of 104 kDa with a PDI of 1.90,

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and DPP-DT-L had an Mn of 15 kDa with a PDI of 4.19. Due to the alternating donoracceptor backbones, DPP-DT polymers showed broad absorption from 600 to 850 nm in chloroform solution, as shown in Fig. 2a. The MWs and photophysical parameters are summarized in Table 1. The peak mass extinction coefficient (772 nm) of DPP-DT-H was

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determined to be 81.7 L g-1 cm-1, which was 1.2-fold larger than that (756 nm) of DPP-DT-L (66.3 L g-1 cm-1), but was 4.1-fold larger than that of gold nanorods (GNRs, 20 L g-1 cm-1) [49]. Furthermore, this value was also much higher than that of many previously reported

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photothermal agents, e.g., 5.94 L g-1 cm-1 for graphene oxide [28], 14.8 L g-1 cm-1 for black phosphorus quantum dots [50], 21.1 L g-1 cm-1 for reduced graphene oxide (rGO) [51], 23.8 L

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g-1 cm-1 for WS2 [52], and 28.4 L g-1 cm-1 for MoS2 nanosheets [29], Moreover, DPP-DT-H polymers showed almost no fluorescence under 808 nm excitation, indicating that the excited states were dominantly relaxed via non-radiative decay.

2.2 Tunable NIR absorption of polymer nanoparticles Semiconducting polymer nanoparticles were prepared via a nanoprecipitation approach by folding and distorting the polymer backbone through hydrophobic interactions. The sizes 6

ACCEPTED MANUSCRIPT of the DPP-DT nanoparticles could be fine-tuned by controlling the starting polymer concentration. Intriguingly, we found that the optical absorption of the DPP-DT nanoparticles was highly dependent on the particle size (Fig. 2b–i). In the case of the DPP-DT-H polymer, the absorption peak was tuned from 691 nm to 811 nm by simply increasing the particle size

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from 13 nm to 255 nm (Fig. 2b). Combined with the low-MW polymers, the resulting nanoparticles with different sizes and MWs showed different NIR absorption peaks from 630 nm to 811 nm and their main absorption bands (above half-maximum) varied from 559 nm to

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934 nm (Fig. 2c, Table 1). The particle size and morphologies of these particles were characterized by dynamic light scattering (DLS) and transmission electron microscopy

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(TEM), as shown in Fig. 2d–i. The observation of blue-shifted absorption with decreasing particle size is consistent with an overall decrease in the conjugation length, attributable to the bending or kinking of the polymer backbone. Although inorganic nanomaterials such as quantum dots and gold nanostructures have already been extensively studied to evaluate their

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size-dependent optical properties [53-55], to the best of our knowledge, this is the first report showing that organic semiconducting polymer nanoparticles have size-dependent properties with tunable absorption in excess of 100 nm. The ability to tune the wavelength and

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magnitude of absorption without changing the chemical composition makes these organic nanomaterials particularly useful for organic optoelectronic devices and biomedical

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applications.

The Pdots were further functionalized with PEG groups through a simple nanoprecipitation method using an amphiphilic polymer, polystyrene-grafted ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH) as the encapsulation matrix (Fig. 3a) [56]. The zeta potentials, average diameters, and morphologies of the Pdots were determined by DLS and TEM measurements. The DLS results and TEM imaging showed that the PEGylated Pdots had uniform spherical shapes (Fig. 3c), with a hydrodynamic diameter of 24 nm (Fig. 3b) and a zeta potential of −35 mV (Fig. S6, Supporting Information). These Pdot 7

ACCEPTED MANUSCRIPT solutions remained stable for months at room temperature with no obvious signs of further aggregation (Fig. S7, Supporting Information). Moreover, the Pdots also show a good colloidal stability in phosphate buffered saline (pH 7.4) and serum-containing culture medium ranging around 25 nm and 28 nm, respectively (Fig. S8, Supporting Information). No obvious

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changes in size and aggregation were observed for 24 h, indicative of the long term colloidal stability in physiological relevant fluids. In fact, the Pdots in different physiological conditions were examined in our previous reports [8, 56]. BSA-passivated Pdots were

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colloidally stable for six months.

The PEGylated DPP-DT-H Pdots showed higher light-harvesting efficiency and thermal

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stability. The photothermal effects of DPP-DT-H Pdots were also evaluated upon continuous laser irradiation at 808 nm. The results revealed that the DPP-DT-H Pdots possessed very high photothermal conversion efficiencies. The temperature elevation of the DPP-DT-H solution (0.05 mg mL−1) could reach as high as 62°C under the NIR laser at a low power

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density of 0.5 W cm-2 for 10 min (Fig. 3e). As expected, such temperature evolution was strongly dependent on the concentration (Fig. 3d, f). With fine control of temperature elevation, the DPP-DT-H Pdots became flexible and efficient photothermal agents for cancer

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cell killing and tumor ablation. To further evaluate the photothermal stability of the DPP-DTH Pdots, the temperature of the DPP-DT-H Pdot solution upon NIR laser irradiation at 808

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nm with reversible heating (laser on) and cooling (laser off) was monitored over time. The recyclable temperature evolution in the presence of the DPP-DT-H Pdots is illustrated in Fig. 3g. During five cycles, the photothermal activity of the DPP-DT-H Pdots did not obviously deteriorate. Noteworthy is that the size and morphology of DPP-DT-H Pdots with no obvious change after 1 h continuous NIR laser irradiation at a power density of 0.5 W cm-2 (Fig. S9, Supporting Information), demonstrating good photostability of DPP-DT-H Pdots. Moreover, the photothermal conversion efficiency (η) value of the DPP-DT-H Pdots was calculated as 55% (Fig. S10 and Equation S1–S9, Supporting Information), according to a previously 8

ACCEPTED MANUSCRIPT reported method [49, 57]. The large absorption coefficient and the high photothermal conversion efficiency of the DPP-DT-H Pdots indicate their great potential for PTT applications. Accordingly, the PEGylated DPP-DT-H Pdots were used for subsequent in vitro

2.3 In vitro NIR photothermal killing of cancer cells

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and in vivo photothermal experiments.

We first investigated the biocompatibility of Pdots before using them as a therapeutic agent

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for PAI and PTT treatment. The in vitro potential cytotoxicity was assessed using standard 3-

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[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays at 24 h and 48 h. No obvious toxic effects (cell viabilities > 90%) were observed for the Pdots using both MCF-7 breast cancer cells and HeLa cells in a broad concentration range of 0–200 µg mL-1, indicating that these Pdots had no obvious toxicity in this concentration range (Fig. 4a, b). However, the cell viability decreased significantly after exposure to NIR laser irradiation for

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in vitro cancer cell killing. The growth inhibition of cells was strongly dependent on the Pdot concentration and the power density of the 808-nm laser. To explore the photothermal efficiency, the Pdots were added to the cell culture medium at various concentrations. After

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incubation of MCF-7 cells with the Pdots for 6 h, the cells were then exposed to the 808-nm

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NIR laser at different laser power densities for 5 min. The results of viability analysis showed that MCF-7 cells were killed in a concentration-dependent manner after NIR laser-induced photothermal effects with the Pdots as photothermal agents (Fig. 4c). Obvious damage to cancer cells was observed after incubation with 50 µg mL-1 Pdots and irradiation under a lowdensity NIR laser at 0.5 W cm-2 with a short irradiation time of 5 min. All of these PTT conditions were milder than those of most previously reported photothermal agents. In additional experiments, we also stained the cells with a live/dead cell double-staining kit containing the acetoxymethyl ester of calcein (calcein-AM, stains viable cells with green 9

ACCEPTED MANUSCRIPT fluorescence) and propidium iodide (PI, stains dead cell with red fluorescence) to monitor viable and dead cells with a confocal fluorescence microscope (Fig. 4d–h). Fluorescence imaging of the stained MCF-7 cells under NIR laser treatment with different power densities demonstrated the high efficacy of the Pdot-mediated photothermal ablation of cancer cells in

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vitro.

2.4 In vivo PAI of tumors

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Motivated by the high in vitro PTT efficacy of the Pdots, we next investigated the feasibility

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of using the Pdots as a unique therapeutic agent for in vivo PAI and PTT in a H22 (and/or 4T1) tumor model (Fig. 1b). Thermal imaging with an infrared thermal mapping apparatus was used to monitor the temperature changes in the tumor area in different groups. For the treatment group, the mice were intratumorally injected with the Pdots. The results showed that the local tumor temperature rose rapidly by about 26°C within 5 min under irradiation with an

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808-nm laser irradiation at a power density of 0.5 W cm-2 (Fig. 5a, b). The temperature was elevated inside the tumor sites, owing to the superior spatial and temporal control of the 808nm laser. In comparison, the local tumor temperature with phosphate-buffered saline (PBS)

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under the same irradiation conditions showed a very minor temperature elevation, which was

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approximately 1/6 that of the tumor with Pdots (Fig. 5f). In the other control groups, the temperature of mouse tumors was maintained at a constant level for the PBS and Pdots groups without laser irradiation. Previous studies have shown that a temperature increase by about 20°C is sufficient to induce thermal damage in the cells in the identified tissue zone with irreversible tissue injury [35], implying that the H22 tumors could be efficiently ablated under these conditions. PAI was then employed to evaluate the photoacoustic properties of the Pdots. The PA spectra of Pdots were acquired at the same mass concentration (0.25 mg/mL) of DPP-DT-H 10

ACCEPTED MANUSCRIPT by pulsed laser irradiation ranging from 680 to 900 nm (Fig. S11, Supporting Information). The PA amplitudes of Pdots at 700 nm were determined at a series of concentrations. As show in Fig. S12, DPP-DT-H Pdots displayed a linear relationship between PA signal and concentration. A photoacoustic microscopy (PAM) system (Fig. 5d) was applied for the in

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vivo imaging of tumors with saline or DPP-DT-H Pdots. The light source of the PAM system was supplied by an OPO laser (Surelite II-20; Continuum, Santa Clara, CA, USA) with a 20Hz pulse repetition rate. A laser beam with a wavelength of 700 nm was equally divided and

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coupled into two optical fibers and then directed onto the sample for photoacoustic signal generation. The incident laser pulse energy was ~10 mJ cm−2, which was well below the

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American National Standards Institute (ANSI) safety standard of 20 mJ cm−2. A focused single-element ultrasound transducer with a 7.5-MHz central frequency was employed in this experiment and coaxially aligned with the laser-focused point. The mice were anesthetized by inhalation with 2% isoflurane. Then, the mice were placed underneath a thin clear plastic

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membrane of a water tank with a circular (8 cm in diameter) opening. Laser-produced acoustic pressure was detected by the ultrasonic transducer, amplified by the pulser/receiver, and then transferred to a high-precision data acquisition card in the computer for further post-

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processing. A computer was utilized to control the two-dimensional translation stage for raster scanning of the subject through the opening.

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The in vivo 3D PAI of tumors was obtained using this PAM system. Fifty layers of PAM scanning were established to completely cover the tumors, which also enabled construction of 3D images of the tumors. As shown in Fig. 5c, the location of the tumor could be detected in both 3D PAM images, and the photoacoustic signal intensity of tumors with Pdots was obviously higher than that of tumors with saline. Notably, 700-nm light is in the optical absorption window in which the tissue itself can emit a low level of photoacoustic signals. In contrast, tumors with Pdots have significant photoacoustic signal enhancement caused by the strong absorption of the Pdots at 700 nm. Orthogonal cross-sections were extracted from each 11

ACCEPTED MANUSCRIPT 3D image along the red dashed lines to provide clear characterization of tumors from the internal view. The maximum PAI intensity in the tumor region was extracted from each layer and calculated to enable a quantitative comparison. As shown in Fig. 5e, the maximum photoacoustic signal intensity of tumors with Pdots was 85.6 ± 6.3 (mean ± standard

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deviation), which was approximately 2.6-fold greater than that of the tumor with saline (33.4 ± 5.2).

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2.5 In vivo photothermal ablation therapy

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We further investigated the PTT efficiency of the Pdots under NIR irradiation of 808 nm in vivo using female Institute of Cancer Research (ICR) mice. When the tumors in female ICR mice reached a size of about 120 mm3, the mice were divided into four groups (five mice per group). For the treatment groups, the tumors on mice with intratumoral (i.t.) injection of the Pdots (1.0 mg kg-1) were irradiated under the 808-nm laser at 0.5 W cm-2 for 5 min. Pdots

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were trapped in the tumor after i.t. due to the enhanced permeability and retention (EPR) effect of tumor tissues [58]. However, Pdots have similar dynamic biodistribution in major organs such as liver and spleen [59, 60]. Mice in other control groups included those injected

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with PBS or the Pdots without NIR laser irradiation and those with PBS injection upon NIR

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laser irradiation (Fig. 6a). The tumor size and weight of the mice were monitored every other day after treatment (Fig. 6b). The results indicated that only the tumors injected with PEGylated DPP-DT-H Pdots and subsequently exposed to the 808-nm laser at 0.5 W cm-2 could be greatly inhibited, leaving black scars at the original tumor sites; these scars faded in about 10 days. In contrast, mice in the control groups demonstrated analogous tumor growth ratios, indicating that neither laser irradiation at 0.5 W cm-2 nor the Pdot injection alone could hinder tumor growth (Fig. 6a, Fig. S13, Supporting Information). Notably, mice harboring H22 tumors subjected to the Pdot-induced PTT treatment were tumor-free (Fig. S14, 12

ACCEPTED MANUSCRIPT Supporting Information) and survived for over 60 days (Fig. 6e) without tumor recurrence. In contrast, mice in the other three control groups showed an average life span of 16–28 days (Fig. 6c), further demonstrating the excellent PPT efficacy of the Pdots. Moreover, neither death nor obvious body weight variations in mice were noted after injection of the Pdots at a

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dose of 1.0 mg kg-1 and PTT treatment (Fig. 6f). Finally, to better elucidate the therapeutic effects of the Pdots during PTT, hematoxylin and eosin (H&E)-stained sections from the liver, spleen, kidney, heart, and lungs of the mice in these groups (Fig. 6g, Fig. S15, Supporting

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Information) were further examined. No appreciable signs of organ damage or inflammatory lesions were observed, suggesting low toxicity to the major organs. Overall, our results

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indicated that the Pdots had superior theranostic capability for PTT of tumors with excellent biocompatibility.

3. Conclusion

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In summary, we developed a series of highly absorbing NIR Pdots for combined PAI and PTT theranostic applications. Intriguingly, for the first time, we found that these organic semiconducting polymer nanoparticles had different NIR absorption peaks after alterations in

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polymer MW and particle size. The Pdots showed high mass extinction coefficients and high

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non-radiative decay rates, resulting in a relatively high photothermal conversion efficiency. Thus, these materials may serve as promising PTT and PAI agents without inducing obvious side effects for in vivo imaging-guided photothermal cancer treatment. Therefore, this safe and effective theranostic platform based on semiconducting Pdots may have further clinical applications in cancer treatment.

4. Experimental Section 4.1. Materials 13

ACCEPTED MANUSCRIPT All materials were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification unless otherwise indicated. Tetrakis(triphenylphosphine) palladium(0) (Pd[PPh3]4, 99%) and chloroform-d were obtained from J&K Chemical Ltd. (Beijing, China). 3,6-Bis(5-trimethylstannylthien-2-thienyl)-2,5-bis(2-decyltetradecyl)-2,5-dihydropyrrolo[3,4-

Optoelectronic

5,5'-dibromo-2,2'-dithiophene were obtained

Materials

Science

&

Technology

Co.,

from

Derthon

(Shenzhen,

China).

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c]pyrrole-1,4-dione and

Ltd.

Tetrahydrofuran (THF) was used to prepare the Pdots and was pretreated with sodium,

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followed by distillation. Ultrapure H2O (18.25 MΩ•cm−2 at 25°C) was used throughout the

4.2. Characterization methods

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study, and all other chemical reagents were used as received.

Ultraviolet (UV)-Vis-NIR absorption spectra were recorded on a Shimadzu UV-2550 spectrophotometer. The average particle size, diameter distributions, and zeta potential of the

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Pdots were determined by DLS with a Malvern Zetasizer Nano ZS instrument. The sizes and morphologies of the Pdots were investigated using TEM (H-600; Hitachi, Japan). NMR spectra were recorded on a Varian Mercury 300 NMR spectrometer (300 MHz for 1H,

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referenced to TMS at δ = 0.00 ppm). GPC measurements were performed on a 515HPLC

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pump (Waters, Milford, MA, USA), with a 2414 refractive index detector. MTT assays were performed using a microplate reader (BioTek Cytation 3, BioTek, Winooski, VT, USA). Fluorescence imaging was performed on a confocal laser scanning microscope (Olympus FV1000).

4.3. Synthesis of DPP-DT with different MWs The polymer DPP-DT was synthesized through a combination of donor-acceptor moieties by Stille cross-coupling polymerization as shown in Scheme 1. In a 25-mL round14

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flask,

3,6-bis(5-trimethylstannylthien-2-thienyl)-2,5-bis(2-decyltetradecyl)-2,5-

dihydropyrrolo[3,4-c]pyrrole-1,4-dione (monomer 1, 325 mg, 0.25 mmol) and 5,5'-dibromo2,2'-dithiophene (monomer 2, 85 mg, 0.25 mmol) were dissolved in toluene (10 mL). The reactants were then degassed and recharged with three freeze-pump-thaw cycles to remove air

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before and after adding the palladium catalyst Pd(PPh3)4 (5 mg, 0.004 mmol). The reactants were stirred at 100°C for different durations (24, 48, or 72 h) under a nitrogen atmosphere. (4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzene (20 mg) and bromobenzene (0.2 mL)

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were then gradually added to remove the end groups separately. Once the hot solution reached room temperature, the solution was dropped slowly into methanol (150 mL). The precipitates

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were collected through filter paper and washed with ultrapure water. The crude polymer (dark solids) was purified using Soxhlet extraction with methanol and acetone to remove low-MW species and catalyst residues for 48 h. The DPP-TH crude product was dissolved in chloroform, filtered with a 0.22-µm membrane, and reprecipitated in methanol (100 mL). The

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resulting polymer was collected by filtration and then dried at 40°C under a vacuum for 2 days, finally yielding a dark solid (374.0 mg, 81%): 1H NMR (300 MHz, CDCl3, δ): 8.95 (br, 2H), 7.50–6.41 (br, 6H), 4.01 (br, 4H), 2.10–0.72 (br, 94 H). MW was measured by GPC as

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Mn = 153354, Mw = 263961, PDI = 1.72.

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4.4. Preparation of DPP-DT nanoparticles with different sizes NIR-absorbing DPP-DT nanoparticles were prepared using the nanoprecipitation method [7]. In a typical preparation, the conjugated polymer DPP-DT was dissolved in anhydrous THF at various concentrations from 0.01 to 2 mg mL-1. A 1-mL aliquot of the above solution was then quickly injected into 5 mL of ultrapure water under vigorous sonication. THF was then evaporated from the suspension by nitrogen purging. Nanoparticles with larger sizes were obtained when the concentration of the initial polymer solution was increased. 15

ACCEPTED MANUSCRIPT 4.5. Preparation of PEGylated DPP-DT Pdots Pdots were prepared according to the nanocoprecipitation method, as described previously [56]. In a typical preparation, THF solution (5 mL) containing DPP-DT (0.8 mg)

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and PS-PEG-COOH (0.2 mg) was quickly dispersed into 10 mL ultrapure water under vigorous sonication. The THF was then removed by nitrogen bubbling on a hotplate. A small fraction of aggregates was removed by filtration through a 0.22-µm membrane filter.

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4.6. Cell culture and cytotoxicity assay

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MCF-7 breast cancer cells and HeLa cervical cancer cells were cultured at 37°C in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and 1% penicillin/streptomycin with a humidified environment containing 5% CO2. The activities of both cell types in vitro were evaluated using MTT assays. MCF-7 cells and HeLa cells were

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seeded into U-bottom 96-well cell culture plates (Costar, Chicago, IL, USA) at a density of 5 × 104 cells well -1 until adherent. The medium was then replaced with DPP-DT-H Pdots at various mass concentrations (0–200 µg mL-1), and the cancer cells were incubated at 37°C in

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an atmosphere containing 5% CO2 for 24 h or 48 h. Subsequently, MTT (20 µL, 5 mg mL−1) was added to each well, and samples were incubated for an additional 4 h. After the addition

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of dimethyl sulfoxide (150 µL well−1), a microplate reader (BioTek Cytation 3) was used to measure the absorbance value (OD570) of each well with background subtraction. The viability of cells was calculated according to the following equation: Cell viability ( % ) =

Where

At × 100% Ac

(1)

is the mean absorbance of the treatment group and

control group.

16

is the mean absorbance of the

ACCEPTED MANUSCRIPT 4.7. In vitro PTT experiments For the PTT experiments, MCF-7 cancer cells were seeded into U-bottom 96-well cell culture plates at 5 × 104 cells well-1 until adherent and then incubated with various mass concentrations of DPP-DT-H Pdots. The cells were treated with an 808-nm laser at a power

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density of 0.5 W cm-2 or 0.75 W cm-2 for 5 min. The relative cell activities were evaluated by standard MTT assays. Cell viability was expressed by the ratio of absolute absorbance of the cells incubated with DPP-DT-H Pdots to that of the cells incubated with culture medium only.

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For fluorescence imaging, MCF-7 cancer cells after irradiation by the 808-nm laser at

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different power densities for 5 min were stained using a live/dead cell double-staining kit (containing calcein-AM and PI), and then imaged using a laser-scanning confocal microscope. 4.8. Tumor mouse model

All experiments involving animals were carried out in accordance with the Guidelines stipulated by the Animal Care and Use Committee (IACUC), University of Macau. Female

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ICR mice were obtained from Beijing HFK Bioscience Co., Ltd. and used at 6 weeks of age. The H22 tumor model was generated by subcutaneous injection of H22 hepatoma ascites in a

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volume of 100 µL into the dorsal area of each female ICR mouse. 4T1 tumor models were generated by subcutaneous injection of 7 × 107 cells in 50 µL PBS into the flanks of the

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female nude mice.

4.9. In vivo PAI of tumor

Nude mice bearing 4T1 tumors were administered DPP-DT-H Pdots (0.2 mg/mL, 50 µL) or saline solution (50 µL, as contrast) through i.t. injection. Twelve hours later, the mice were anesthetized by inhalation of 2% isoflurane and were restrained in a holder designed specifically for PAI [61]. The photoacoustic images were recorded with a PAM with a laser at a wavelength of 700 nm. 17

ACCEPTED MANUSCRIPT 4.10. In vivo PAI and PTT ICR mice bearing H22 tumors were randomized into four groups after the tumors xenografts reached ∼120 mm3 (n = 5 per group): (i) i.t. injection of PBS (60 µL); (ii) i.t.

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injection of PBS (60 µL), irradiated with an NIR laser (808 nm, 0.5 W cm-2, 5 min); (iii) i.t. injection with DPP-DT-H Pdots (60 µL, 0.5 mg mL-1); and (iv) i.t. injection with DPP-DT-H Pdots (60 µL, 0.5 mg mL-1), irradiated with an NIR laser (808 nm, 0.5 W cm-2, 5 min). The

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mice were anesthetized by inhalation of 2% isoflurane, and the temperature changes at the tumor sites were monitored using a FLIR E8 thermal imaging camera (FLIR Systems, Inc.,

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USA). The tumor sizes were measured using digital calipers at regular intervals. The volume was calculated according to the following equation:

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4.11. Histopathological evaluation

(2)

For histology analysis, the harvested organs were fixed in 4% neutral buffered paraformaldehyde and embedded with paraffin. Sections of the main organs of interest

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(including the heart, liver, spleen, lung, and kidney) of the mice were stained with H&E. The

Notes

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histological sections were imaged using an optical microscope.

The authors declare no competing financial interest.

Acknowledgements C. Wu acknowledges financial support from “Thousand Young Talents Program” and National Science Foundation of China (Grants 61222508 and 61335001), The study was also 18

ACCEPTED MANUSCRIPT supported in part by Grants MYRG2014-00093-FHS, and MYRG 2015-00036-FHS from the University of Macau and FDCT Grants 026/2014/A1, 025/2015/A1, and 052/2015/A2 from the Macao government.

Supplementary data related to this article can be found at

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https://www.journals.elsevier.com/biomaterials.

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Appendix A. Supplementary data

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Table 1. Summary of Molecular Weights and Photophysical Properties of DPP-DT Polymers

DPP-DT-M

a)

104

153

63

198

264

c)

PDI

abs

λmax d) (nm)

εmax

705

65.0

756

66.3

e)

4.19

1.90

1.72

758

73.8

772

Number-average molecular weight;

abs

Size f) (d,nm)

λmax g) (nm)

13

630

43

651

164

687

37

701

91

709, 758

142

712, 772

78

727, 789

122

795

220

808

255

811

81.7

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DPP-DT-H

15

Mw b) (kDa)

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DPP-DT-L

Mn a) (kDa)

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Copolymers

b)

Weight-average molecular weight;

c)

Polydispersity

index; d)Absorption maximum in chloroform; e)Mass extinction coefficient; f)Size of DPP-DT

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in water-dispersible NPs; g)Absorption maximum of DPP-DT NPs.

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Fig. 1. (a) Synthetic route and chemical structure of the conjugated polymer DPP-DT via

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Stille polymerization. (b) Schematic illustration of PEGylated DPP-DT Pdots as a

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photoacoustic imaging and photothermal therapy agent.

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Fig. 2. (a) Absorption of DPP-DT and GNR. The DPP-DT polymers had number-average molecular weights (Mn values) of 15, 104, and 153 kDa. (b) Absorption spectra (plotted from left to right with peaks at 691, 704, 715, 778, 795, and 811 nm) of DPP-DT-H nanoparticles

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with corresponding hydrodynamic particle sizes of 13 (black), 28 (red), 51 (olive), 122 (royal blue), and 255 nm (purple). The dotted lines represent the absorption spectra of DPP-DT-H in chloroform. (c) Absorption spectra of DPP-DT nanoparticles with different Mn values and

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particle sizes (see Table 1 for details). (d) Size distribution histogram of DPP-DT nanoparticles by DLS. (e–i) TEM images of DPP-DT-H nanoparticles of different sizes.

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Fig. 3. (a) Schematic illustration showing the fabrication process of PEGylated DPP-DT-H Pdots. (b) Hydrodynamic sizes of PEGylated DPP-DT-H Pdots measured by dynamic light

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scattering. (c) TEM images of DPP-DT-H Pdots. (d) Photos of DPP-DT-H Pdots with different concentrations. (e) Photothermal imaging was carried out by monitoring DPP-DT-H

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Pdot solutions (50 µg mL−1) irradiated using an 808-nm NIR laser. (f) The temperature evolution of DPP-DT-H Pdots with various Pdot concentrations under 808-nm laser irradiation at a power density of 0.5 W cm-2. (g) Temperature curves of DPP-DT-H Pdots and water under five cycles of photothermal heating by an 808-nm laser.

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Fig. 4. Relative cell viabilities of MCF-7 cells (a) and Hela cells (b) incubated with PEGylated DPP-DT-H Pdots at various concentrations for 24 h or 48 h. (c) Relative viabilities of MCF-7 cells incubated with various concentrations of PEGylated DPP-DT-H Pdots with laser irradiation at 808 nm (0.5 and 0.75 W cm-2) for 5 min. (d–h) Confocal fluorescence images of PEGylated DPP-DT-H Pdots (50 µg mL-1) incubated with MCF-7 cancer cells after

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irradiation by the 808-nm laser at different power densities for 5 min. The cells were

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costained with calcein-AM and propidium iodide before imaging. Scale bar: 200 µm.

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Fig. 5. (a) IR thermal images of H22 tumor-bearing mice after injection of PBS or PEGylated

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DPP-DT-H Pdots (1 mg kg-1) with laser irradiation (808 nm, 0.5 W cm-2) over time. (b) Heat curves of tumors in mice treated under different conditions. The four groups are plotted with different colors: PBS (black); PBS, irradiated using an NIR laser (blue); DPP-DT-H Pdots without laser irradiation (magenta); DPP-DT Pdots, irradiated using an NIR laser (red). (c) In vivo 2D and 3D photoacoustic images of tumor tissues with injection of saline or DPP-DT-H Pdots. (d) Schematic of our homemade multispectral photoacoustic and ultrasonic microscopy system. (e) Photoacoustic intensities of tumor tissues following the intratumoral administration of DPP-DT-H Pdots. (f) Maximum temperature differences in tumor tissues following the intratumoral administration of DPP-DT-H Pdots after irradiation with an 808nm laser at 0.5 W cm-2 for 5 min. 32

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Fig. 6. Investigation of synergistic cancer therapy in vivo. (a) Photographs documenting H22 tumor development at different days in live mice treated under different conditions. The four groups are plotted as follows from left to right: (i) PBS; (ii) PBS, irradiated using an 808-nm laser (0.5 W cm−2); (iii) Pdots; (iv) Pdots, irradiated using an 808-nm laser (0.5 W cm−2). (b) Growth curves of H22 tumors on mice of different groups after corresponding treatment as indicated. Data points represent the means ± standard deviations of five mice per group. (c) Survival curves of various groups of tumor-bearing mice after different treatments. (d) Photographs of tumors after excision from different groups of mice at the end of treatment. (e) Survival of Pdot-injected mice after PTT treatment over 60 days. (f) Body weight changes were recorded after therapy every 2 days. (g) H&E-stained images of the major organs from healthy control mice and DPP-DT-H Pdot-injected mice 60 days after PTT treatment. Scale bar: 200 µm. 33

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Graphical abstract

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