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Simple D-A-D Structural Bisbithiophenyl Diketopyrrolopyrrole (TDPP) as Efficient Bioimaging and Photothermal Agents shan zong, xin wang, Wenhai Lin, Shi Liu, and Wei Zhang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00333 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Simple

D-A-D

Diketopyrrolopyrrole

Structural (TDPP)

Bisbithiophenyl as

Efficient

Bioimaging and Photothermal Agents Shan Zong,a Xin Wang,b Wenhai Lin,c,d Shi Liu*c,† and Wei Zhang*,c,d a b

The First Hospital of Jilin University, Changchun 130021, China Department of Thyroid Surgery, The First Hospital of Jilin University, 71 Xinmin

Street, Changchun, Jilin 130021, PR China c

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of

Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China d

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

*

corresponding authors: [email protected]; [email protected]

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Design and synthesis of biocompatible and multi-functional photothermal agents is crucial for effective cancer phototherapy. In order to achieve this ambition, simple D-A-D structural bisbithiophenyl diketopyrrolopyrrole (TDPP) was fabricated. In this molecule, the donor, 2-thiophenylboric acid, was conjugated via Suzuki coupling reaction, which could expand the emission wavelength to the red region of the spectrum. TDPP could self-assemble into stable and uniform nanoparticles (TDPP NPs) in assistant of amphiphilic Pluronic F-127 polymer. Exposing TDPP NPs (100 µg/mL) aqueous dispersion to 638 nm (0.61 W/cm2) laser irradiation resulted in a temperature elevation of approximately 30 oC within 5 min, which is high enough for inducing the cytotoxicity and tumor inhibition. Because of the bathochromic shift absorption of TDPP NPs in water, TDPP NPs could also act as a contrast agent for near-infrared fluorescence imaging (NIRF) to visualize the drug distribution in vivo. Coupled with the infrared thermal imaging properties of the photothermal agent, TDPP NPs were proved to be a multifunctional theranostic agent for dual-modal imaging-guided phototherapy.


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INTRODUCTION

In recent years, cancer has become one of the worldwide leading killers which causes majority of human death.1-3 Presently, the therapeutic methods of tumors clinically could be divided into surgery treatment, radiotherapy and chemotherapy. However, surgery in many occasions is not able to completely remove all cancer cells in the human body, chemotherapy and radiotherapy suffer from their serious toxic side effects to normal tissues and limited specificities to cancer cells.4 Phototherapies, as a burgeoning cancer treatment, have attracted more and more attention due to its controllability and low invasiveness.5, 6, 7 During phototherapies process, a preferable wavelength light source, especially near-infrared (NIR) light with deep-enough tissue penetration ability, and effective photoabsorbing agents with negligible dark cellular toxicity and obvious cytotoxicity to cause cellular death upon irradiation. Phototherapies include photodynamic therapy (PDT) and photothermal therapy (PTT).8-14 Different from PDT therapy, which is oxygen-dependent treatment, PTT is a process which could convert the incoming light to generate localized heat for causing irreversible death of the targeted tumor cells.15 In the past few decades, numerous of photothermal agents including both inorganic and organic materials were widely explored, which have achieved encouraging therapeutic effects in preclinical animal experiments.16-20 Among these materials, 1,4-diketo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DPP) based photothermal agents, which were compromised of a π-conjugated core and two alkyl chains with quaternary ammionium, have gradually attracted much attention.21 As far as we known, DPP have been well studied as OLEDs, solar cells, fluorescent probes and two-photon absorption materials due to their brilliant light, humidity, and heat stability, which could expand the emission wavelength of dyes greatly.22-27 In recent years, DPP have been used as PTT agents especially after being incorporated into donor-acceptor-donor (D-A-D) systems, such as the semiconducting polymer nanoparticles (SPNs).28-36 In solution, isolated DPP molecules tend to exhibit relatively high fluorescence quantum yields, however, the fluorescence quenched efficiently after self-aggregation, which endowed DPP with excellent photothermal conversion efficiency (η).37-40 While, most of the DPP-containing PTT agents required relatively complex synthesis, and ingenious design of molecular structure. Therefore, it is imperative to fabricate DPP-based materials with simple D-A framework structure and high conversion efficiency.



In this article, we have synthesized an organic molecule, bisbithiophenyl diketopyrrolopyrrole (TDPP) with typical D-A-D structure. In order to obtain uniform and stable nanoparticles with high drug loading efficiency, we used amphiphilic Pluronic F-127 to fabricate the TDPP@F-127 NPs (TDPP NPs). TDPP NPs exhibited effective photothermal treatment capability. Both in vitro and in vivo studies confirmed the good biosafety and high anti-tumor efficiency of TDPP NPs under laser irradiation, and TDPP NPs could serve as multifunctional theranostic agents for imaging guided photothermal therapy (Scheme 1).

Scheme 1. Schematic presentation of preparation and application of TDPP NPs in dual-modal imaging guided photothermal therapy.


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RESULTS Preparation and Characterization of TDPP NPs. Firstly, the thiophen-substituted DPP (thiophen-diketopyrrolopyrrole, TDPP) was synthesized by traditional Suzuki coupling reaction.41, 42

After purification on silica gel chromatography, TDPP was obtained and then characterized by

proton nuclear magnetic resonance spectroscopy (1H NMR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Figure S1 in

Supporting Information), which validated the successful synthesis of the targeted molecule. In order to obtained the nanoparticles formulation of TDPP, the amphiphilic copolymer Pluronic F-127 was used to facilitate the self-assembling process in aqueous solution. Briefly, a tetrahydrofuron solution of TDPP and F-127 (mass ratio was 1:10) was added dropwise into deionized water under vigorous stirring. After the complete evaporation of THF and dialysis, the TDPP@F-127 NPs (TDPP NPs) were obtained. The content of TDPP in the nanoparticles was determined via the calibration curve of UV-vis. The morphology and size distribution were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). As shown in Figure 1a, the TEM imaging demonstrated that the TDPP NPs showed a spherical morphology with an average size of about 70 nm, which was smaller than that measured via DLS (as shown in Figure 1b, average diameter: 125.6; PDI: 0.235). A possible reason might be that the TEM images were taken under dry formulation. Subsequently, the colloidal stability was monitored by the changes of size and size distribution was monitored in water, phosphate buffer saline (PBS, pH 7.4) containing 10% fetal bovine serum (FBS) and cell culture medium (Dulbecco’s modified Eagle’s medium, DMEM) for different period. Negligible changes were found through the results monitored by the DLS, which indicated the favorable stability of the NPs (Figure 1c and 1d).



Figure 1. (a) TEM images of TDPP NPs, scale bar, 500 nm. (b) Size distribution results of TDPP NPs measured by DLS in aqueous solution. The changes of the diameter and PDI of the nanoparticles in (c) aqueous solution and (d) in PBS (pH 7.4) containing 10% FBS and DMEM with different times determined by DLS.

Photochemical Spectra of TDPP NPs. The photochemical properties of TDPP NPs and the small organic molecules (TDPP) were studied by UV-vis absorption and photoluminescence spectra. As shown in Figure 2a, TDPP showed two absorption peaks at 565 nm and 604 nm, respectively. Compared with that of the small organic molecules, the the absorption spectra of TDPP NPs in water showed obvious red shifts, which peaked at approximately 589 nm and 645 nm with the increase of the chain length. The bathochromic shift absorption band of TDPP NPs showed possible J-aggregates of the nanoparticles in water, which was similar to the reported phenomenon.39 In Figure 2b, the maximum fluorescence wavelength was seen at 636 nm, however, almost no fluorescence could be seen for TDPP NPs due to the aggregation-caused quenching (ACQ).43 The phenomenon could also been observed visually by photos under the irradiation with 365 nm light. As shown in the pictures in Figure 2b, TDPP in DMF/H2O=1:1 (v/v) emitted strong red fluorescence, while no fluorescence was observed for the nanoparticles in water.


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Figure 2. (a) UV-vis absorption and (b) fluorescence spectrum of TDPP NPs in water and TDPP in DMF. Insets in (b) are photos of TDPP in DMF solution (left) and TDPP NPs in water (right) under natural light and the illumination of UV light (365 nm).

Effect of Photothermal Heating In Vitro. In order to detect the photothermal conversion capability of TDPP NPs, we monitored the temperature changes (∆T) upon laser irradiation. Firstly, temperature increasing of TDPP NPs with various concentrations (10 to 100 µg/mL) was recorded under 638 nm laser irradiation as a function of time. As demonstrated in Figure 3a, the temperature of pure water increased by about 6 oC under continuous laser irradiation for 5 min. However, with increasing concentration of TDPP (10, 25, 50 and 100 µg/mL), the temperature of the solutions augmented by 14.3-29.9 oC, respectively, indicating the effective photothermal conversion ability of the TDPP NPs. Moreover, the temperature of TDPP NPs solution (100 µg/mL) ascended dramatically as the laser power density increased from 0.38 to 1.05 W/cm2 (Figure 3b). In order to further visualize the warming-up effect, infrared thermographic maps of the corresponding hole of 96 plate containing TDPP NPs were taken during the process. As described in Figure 3e, the surface temperature of the solutions increased rapidly under laser irradiation. Meanwhile, the nanoparticles also indicated concentration and laser power intensity dependent PTT effect. All the results above confirmed that TDPP NPs could generate heat after laser irradiation. Subsequently, in order to calculate the photothermal conversion efficiency (η) of TDPP NPs, the temperature changes of the solution (100 µg/mL, 200 µL) as the function of time was measured under successive irradiation of 638 nm laser (0.61 W/ cm2) (in Figure 3c). After reaching the steady state, the laser was shut off and the temperature of the TDPP NPs was recorded to determine the rate of heat transfer ability from solution to the environment. The calculated photothermal conversion efficiency could reach approximately 20 % under the irradiation of 638 nm laser, which was high enough for effective photothermal therapy (as shown



in Figure S2).44 Moreover, the photothermal stability of TDPP NPs was investigated through multiple cycles of heating and cooling. As shown in Figure 3d, the NPs could maintain the photothermal effect with similar temperature increasing, which is important for repeated PTT treatment. Furthermore, the photostability of TDPP NPs was confirmed by using indocyanine green (ICG) as control. In Figure S3, there was almost no degradation in absorption of the NPs, however, the absorption of ICG decrease significantly after continuous laser irradiation for 10 min. The insets pictures also showed no observed color change for TDPP NPs, while obvious fading from light green to colorless for ICG solution.


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Figure 3. (a) Temperature rise curves of TDPP NPs at different concentrations (10-100 µg/mL) under laser irradiation (638 nm, 0.61 W/cm2, V=200 µL) and (b) Temperature elevation of TDPP



NPs (100 µg/mL, V=200 µL) aqueous solution under different powers (0.38-1.05 W/cm2) as a function of time. (c) Photothermal effect of TDPP NPs (100 µg/mL, V=200 µL) under irradiation of a 638 nm laser (0.61 W/cm2), which was shut off after irradiation for 300 s. (d) Temperature variations of NPs (100 µg/mL, V=200 µL) under 638 nm irradiation at a power density of 0.61 W/cm2 for five light on/off cycles. (e) Thermal images of TDPP NPs (100 µg/mL, V=200 µL) solution on 638 nm laser irradiation of 0.61 W/cm2 under different powers at different times.

Cellular Uptake and In Vitro Cytotoxicity. In order to investigate the cellular internalization of TDPP NPs, confocal laser microscopy (CLSM) was carried out on the human cervical carcinoma HeLa cells. Briefly, the cells were incubated with NPs (TDPP: 20 µg/mL) at 37 oC, and the cell nuclei were stained with 4′,6-diamidion-2-phenylindole (DAPI). As demonstrated in

Figure 4a, obvious red fluorescence could be found in cell plasma, indicating that the nanoparticles could be effectively endocytosed by cancer cells. After the cells were incubated with the nanoparticles at 4 oC, the red fluorescence significantly decreased, which suggested the cellular uptake of the nanoparticles was energy-dependent. Moreover, the way of endocytosis was further investigated by colocalization of TDPP NPs (TDPP: 20 µg/mL) and lysosomes labeled with green fluorescence (Lyso tracker green). In Figure 4b, obvious yellow colocalization area of red fluorescence and green fluorescence from Lyso tracker green, indicating the accumulation of TDPP NPs in lysosomes. In addition, flow cytometry was implemented to further compare and quantify the cell internalization of the nanoparticles. It could be observed that the cellular uptake of the NPs enhanced with the incubation time 0.5 to 4 h (as shown in Figure 4c), demonstrating the time-dependent endocytosis behaviors, which was also consistent with the results obtained from CLSM images.


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Figure 4. (a) Representative CLSM images of HeLa cells after incubation with TDPP NPs (TDPP: 20 µg/mL) for 0.5 h, 2 h, 4 h at 37 oC and 0.5 h at 4 oC, respectively. For each panel, the images from left to right show cell nuclei stained by DAPI (blue), TDPP in cells (red), and overlays of both images. (b) Colocalization images of HeLa cells treated with TDPP NPs for 2 h. The images from left to right show cell nuclei stained by DAPI (blue), Lyso Tracker fluorescence (green) TDPP in cells (red) in lysosomes, and overlays of three images. Scale bar: 20 µm. (c) Flow cytometry analyses of the cellular uptake of TDPP NPs (TDPP: 20 µg/mL) over different time (0.5, 2 and 4 h) at 37 oC.

The in vitro PTT effect of TDPP NPs was examined by the relative cell viabilities using HeLa cells

and

mouse

colon

cancer

CT26

cells

through

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. As shown in Figure

5a&c, no significant toxicity could be observed for the cells treated only with the nanoparticles, even at the concentration of 55 µg/mL. However, the NPs exhibited concentration-dependent cytotoxicity upon laser irradiation and the cell survival rate was less than 20% for both HeLa and CT26 cells, validating the remarkable PTT effect of TDPP NPs. In addition, we also compared the



PTT effect of the NPs under laser irradiation with different power intensity. As demonstrated from the results (Figure 5 b&d), the nanoparticles also exhibited power intensity dependent cytotoxicity simultaneously. To further visually demonstrate the cell apotosis and necrosis were caused by PTT treatment, fluorescence imaging was performed by co-staing the cells with calcein AM and propidium iodide to identify live (green fluorescence) and dead/late apoptotic (red fluorescence) cells. Figure 5e showed that most of the cells treated with the NPs in the dark or only exposed to the 635 nm laser were alive with plentiful green fluorescence. Almost all the cells treated with TDPP NPs under irradiation were dead. Moreover, we also treated HeLa cells with multiple concentration drugs and 638 nm laser irradiation with different power intensity. As shown in the pictures in Figure 5e, the nanoparticles revealed concentration and power intensity dependent cellular toxicity. All the results were consistent with the MTT experiments, which indicated TDPP NPs could be served as a promising platform for highly effective PTT in vivo.


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Figure 5. (a) Cell viabilities after incubating with different concentrations of TDPP NPs (6.25-55 µg/mL) wih and without laser irradiation and (b) power intensity dependent cytotoxicity with multiple NPs concentrations of HeLa cells. (c) Cell viabilities after incubating with different concentrations of TDPP NPs (6.25-55 µg/mL) wih and without laser irradiation and (d) power intensity dependent cytotoxicity with multiple NPs concentrations of CT26 cells. (e) Fluorescent images of propidium iodide (red, dead cells) and calcein AM (green, live cells) co-stained HeLa cells after laser irradiation; all the scale bars are 100 µm.



Near Infrared Fluorescence Imaging (NIRF) of TDPP NPs In Vivo. The feasibility of TDPP NPs in vivo near-infrared fluorescence imaging (NIRF) was investigated by using murine cervical cancer (U14 cells) bearing Kunming male mice by intratumor injection of TDPP NPs and then imaged by Meatro 500FL in vivo optical imaging system (Cambridge Research & Instrumentation, Inc. USA) under excitation of yellow light (575-605 nm). Briefly, the normal Kunming mice were used as the controls. Subsequently, the tumor-bearing mice were intratumorly injected of TDPP NPs (1 mg/mL) at the center of the tumor with the depth of insertion about 0.8 cm and then the animals were imaged at different time points. As shown in Figure 6a, the fluorescence emission from the tumor was monitored for about 2 weeks. Only after 30 min after injection, an obvious fluorescence signal was detected at the injection site, and the fluorescence intensity gradually increased and showed a gradient intensity distribution along with different time period. During the whole process, we could detect the distribution of photothermal agents in tumor site via fluorescence imaging method. Then, tumor and the main organs were excised from the mice on the 14th day. As shown in the pictures in Figure 6a, an obvious fluorescence could be observed in tumor site while a weak fluorescence in liver and no fluorescence emission was seen in other organs in 2 weeks after injection. These results indicated that TDPP NPs were retained around the tumor even after 14 days, which is beneficial to maintain the photothermal conversion efficacy at the tumor site. We also demonstrated the imaging capability of TDPP NPs in tumors by tail intravenous injection. In Figure 6b, no fluorescence could be found at tumor site in 0.5 h after injection of TDPP NPs, and the liver showed strong signals. After 6 h, TDPP NPs were observed in tumor and the fluorescence in tumor continuously. However, the most of the injected nanoparticles were mainly accumulated in liver, which was unfavorable for photothermal therapy. Thus, in order to get a higher PTT and optimum treatment efficacy, we decided to use the intratumor injection for further antitumor study of TDPP NPs in vivo.


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Figure 6. (a) In vivo imaging and biodistribution of intratumor injected TDPP NPs in tumor-bearing Kunming male mice at the dose of 1 mg/mL at 0.5 h, 24 h, 48 h, 4 day, 9 day, 14 day, respectively. NIRF images of major organs and tumor after intratumor injection of TDPP NPs at 14th day. 1: tumor, 2: heart, 3: liver, 4: spleen, 5: lung, 6: kidney. (b) In vivo NIRF imaging of the mice after i.v. injected with TDPP NPs (1 mg/mL, V=200 µL) at 0.5 h, 6 h, 24 h, 48 h, 4 day, 9 day, 14 day, respectively. The red circle is the tumor site of the mice. The images on the right side were the changes of the fluorescence intensity of tumor site over time after intratumoral and tail intravenous injection, respectively.

AntitumorEffect of TDPP NPs In Vivo. We proceeded to evaluate the antitumor efficacy in vivo. Briefly, subcutaneous U14 tumors were established in Kunming female mice at the right armpit. When the tumor volume reached 50-100 mm3, mice were randomly divided into four groups: combinations of TDPP NPs and 638 nm laser; TDPP NPs only; 638 nm laser only and the control (PBS) group. TDPP NPs (1 mg/mL, 200 µL) solution was administrated intratumorally in the first two groups, and saline in the control group, respectively. After 2 h post injection, the tumors were exposed to 638 nm laser at 0.61 W/cm2 for 20 min in order to keep the temperature in the tumor area above 45 oC, and the temperature changes of the tumors were recorded by an infrared thermal camera (in Figure 7a). As illustrated in the picture, the temperature of the tumor site of the mice increased gradually under 638 nm laser irradiation (0.61 W/cm2) and the temperature reached about 50 oC after 6 minutes. Moreover, temperature could still maintain at 49



o

C after 20 minutes of continuous illumination, which was high enough to cause the death of

tumor cells in mice. During the whole treatment process, the laser irradiation was carried out only once. The tumor volume and body weight were measured every 2 days in 12 days treatment, then the mice were sacrificed and the tumors were excised. As shown in Figure 7b&c, tumors only injected with saline, laser only and TDPP NPs without laser irradiation grew rapidly during 12 days, indicating no tumor inhibition capability for these groups. However, tumors with the treatment of TDPP NPs with laser irradiation were prominently suppressed and obvious black scars at the original tumor sites were found (the inset picture), which were attributed to the excellent PTT treatment of the nanoparticles. The photos and weight of the excised tumors of various groups were as shown in Figure 7c&S4. It could be observed clearly that the tumors in the TDPP NPs group with laser irradiation were the smallest than that in other groups (in Figure

7d). Hematoxylin andeosin (H&E) staining of tumor regions (Figure S5) further certified the elimination of tumor cells ulteriorly and the remarkable PTT treatment efficacy. Moreover, to evaluate the side effect of TDPP NPs as a PTT agents, the body weight were recorded during the treatment process. As shown in Figure 7e, the body weight of the mice were growing steadily during the treatment process, which indicated the nanoparticles did not cause side effects on normal tissues and the whole body.


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Figure 7. In vivo PTT study. (a) The whole-body photothermal images of the tumor site after intratumoral injection of TDPP NPs solution (0.2 mL, 1 mg/mL) exposure to laser irradiation (638 nm, 0.61 W/cm2) at different time period. (b) Relative tumor volumes of mice treated with PBS, laser only, TDPP NPs only and TDPP NPs (0.2 mL, 1 mg/mL) with laser irradiation. (c) Quantitative analysis of tumor weight of each group. (d) Photos of the excised tumors on the 12th day. (e) Changes in body weight of mice with U14 tumors upon various treatments during 12 days.

CONCLUSION To summary up, a simple and effective photothermal diagnostic agent, bisbithiophenyl diketopyrrolopyrrole (TDPP) has been successfully designed and synthesized. We have demonstrated that the TDPP could form stable and high concentrated nanoparticles (TDPP NPs) in aqueous solution with the help of Pluronic F-127. TDPP NPs demonstrate the capability to be



served as the therapeutic agent for NIRF and photothermal (PT) imaging guided photothermal therapy under irradiation. The dual modal imaging enabled accurate localization of the tumor site in order to avoid unnecessary normal tissue damage, real-time monitoring of the photosensitizer distribution and the temperature change at the tumor site for determining the optimal irradiation protocol. Both in vitro and in vivo studies demonstrated the negligible dark toxicity yet remarkable photothermal treatment effect of TDPP NPs. This work highlights the potential of functional organic molecules and their nanoparticle formulations in biomedical fields.

The Supporting Information is available free of charge.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Project. No. 51522307).

DISCLOSURE STATEMENT The authors report no conflicts of interest

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Simple DAD Structural Bisbithiophenyl ... - ACS Publications

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