Diketopyrrolopyrrole–Triphenylamine Organic Nanoparticles as

Diketopyrrolopyrrole−Triphenylamine Organic Nanoparticles as Multifunctional Reagents for Photoacoustic Imaging-Guided Photodynamic/Photothermal Synergistic Tumor Therapy Yu Cai,† Pingping Liang,† Qianyun Tang,† Xiaoyan Yang,† Weili Si,† Wei Huang,*,† Qi Zhang,*,‡ and Xiaochen Dong*,† †

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), and ‡School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China S Supporting Information *

ABSTRACT: Herein, a donor−acceptor−donor (D-A-D) structured small molecule (DPP-TPA) is designed and synthesized for photoacoustic imaging (PAI) guided photodynamic/photothermal synergistic therapy. In the diketopyrrolopyrrole (DPP) molecule, a thiophene group is contained to increase the intersystem crossing (ISC) ability through the heavy atom effect. Simultaneously, triphenylamine (TPA) is introduced for bathochromic shift absorption as well as charge transport capacity enhancement. After formation of nanoparticles (NPs, ∼76 nm) by reprecipitation, the absorption of DPP-TPA NPs further displays obvious bathochromic-shift with the maximum absorption peak at 660 nm. What’s more, the NPs architecture enhances the D-A-D structure, which greatly increases the charge transport capacity and impels the charge to generate heat by light. DPP-TPA NPs present high photothermal conversion efficiency (η = 34.5%) and excellent singlet oxygen (1O2) generation (ΦΔ = 33.6%) under 660 nm laser irradiation. PAI, with high spatial resolution and deep biotissue penetration, indicates DPP-TPA NPs can rapidly target the tumor sites within 2 h by the enhanced permeability and retention (EPR) effect. Importantly, DPP-TPA NPs could effectively hinder the tumor growth by photodynamic/photothermal synergistic therapy in vivo even at a low dosage (0.2 mg/kg) upon laser irradiation (660 nm 1.0 W/cm2). This study illuminates the photothermal conversion mechanism of small organic NPs and demonstrates the promising application of DPP-TPA NPs in PAI guided phototherapy. KEYWORDS: multifunctional reagents, photoacoustic imaging, phototherapy, organic nanoparticles, tumor

T

Because of the similar light triggering conditions, many efforts have been made to combine PTT and PDT in one treatment system.16−18 Making photosensitizers chlorin e6 (Ce6)19 or indocyanine green (ICG)20 linked to or coated on inorganic photothermal agents (such as gold nanorods,21,22 carbon nanotubes,23,24 two-dimensional nanomaterials25) is one of the common strategies. However, to realize the water solubility, biocompatibility, and photostability, the combination of two or more different components in one system usually requires complicated synthetic routes.26−28 Besides, PDT and PTT

he development of efficient tumor therapies is highly desired as cancer has become one of the major threats to human health. Traditional tumor therapies, such as chemotherapy, radiotherapy, and surgery, usually bring a variety of odious side effects to cancer patients during the treatment.1−3 Currently, mild and noninvasive therapies have attracted extensive attention because of the efficient tumor destruction ability and minimal harm to normal tissues.4−7 Photothermal therapy (PTT)8−10 and photodynamic therapy (PDT)11−13 are two main approaches among the noninvasive therapies for a malignant tumor. For PTT, tumor cells are killed by heat, which is generated by the therapeutic agents under near-infrared (NIR) light irradiation. Differently, for PDT, tumor cells are eliminated by the reactive oxygen species (ROS) generated by photosensitizer under the irradiation.14,15 © 2016 American Chemical Society

Received: November 25, 2016 Accepted: December 29, 2016 Published: December 29, 2016 1054

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Scheme 1. Schematic Illustration of the Enhanced D-A-D Structured DPP-TPA NPs as Theranostic Agents for PAI Guided PDT/PTT

Figure 1. (a) Photographs of DPP-TPA in THF, PBS solution, and DPP-TPA NPs in PBS (pH = 7.4), 60 μg/mL. (b) UV−vis absorption spectra of DPP-TPA in THF and DPP-TPA NPs in PBS (pH = 7.4). (c) TEM image of DPP-TPA NPs. (d) DLS examination for size distribution of DPP-TPA NPs in PBS.

current optical theranostic systems mainly rely on fluorescence. Limited light penetration depth and autofluorescence background greatly compromise the diagnostic accuracy.23,40 Photoacoustic imaging (PAI), a high spatial resolution and deep biotissue penetration imaging technology,41−44 can offer three-dimensional images of tumor tissues in preclinical research and clinical application.45−47 Because the acoustic signal is generated by thermal expansion, many photothermal agents with optical absorption can also be used as photoacoustic contrast agents. Therefore, the design and synthesis of NIR absorbed therapeutic agents to realize PAI-guided PTT/ PDT can simultaneously enhance the efficacy, safety, and accuracy of cancer treatment. Diketopyrrolopyrrole (DPP) derivatives have been widely explored in various electronic devices and fluorescence probe because of their high photostability and planar and conjugated structure.48,49 As a typical electron-deficient core, DPP can attach electron-donating substitutes to form a donor−acceptor−donor (D-A-D) structure, which enhances NIR-absorption and semiconductive property.50,51 And studies also indicate that the charge transport can be obviously facilitated by self-assembly of D-AD molecules.52 Therefore, it is highly essential and desirable to develop D-A-D structured DPP derivatives to explore its PAI

agents combined in one treatment platform often need different excitation wavelengths to produce ROS and heat, respectively, which prolongs the synergistic treatment time and brings more systemic side effects to patients.29,30 Therefore, it is urgent to develop a single component therapeutic agent, achieving photodynamic/photothermal synergistic therapy at a single excitation wavelength. Compared to inorganic therapeutic agents, organic materials possess adjustable absorption wavelength, low-toxicity, good biodegradability, and rapid metabolism in biological tissue.31−33 However, most organic photosensitizers used in PDT still face some major challenges, such as poor water solubility, low photostability, and no tumor targeting in clinic.34−36 Meanwhile, some semiconductor polymers with excellent photothermal conversion efficiency in PTT have the problem of biodegradation.6,31,37 It is well-known that the heat release principle of inorganic NPs is related to the inside mobile electron.38 But the mechanism of photothermal conversion of organic therapeutic agents has not been well explained until now.10 On the other hand, real-time monitoring the delivery of therapeutic agents to the tumor sites offering visible guidance in cancer therapy still is a challenge. So, theranostic integration has been widely investigated in recent years.39 However, 1055

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Figure 2. (a) PA images of DPP-TPA NPs with different concentration in PBS. (b) Relationship between concentration of DPP-TPA NPs and PA intensity. (c) Absorption intensity at 418 nm of DPP-TPA (10−5 mol/L) mixed with DPBF (2 × 10−5 mol/L) in DCM over time at 660 nm laser irradiation. (d) Photothermal profiles of DPP-TPA NPs in PBS under different power density irradiation (660 nm). (e) Photothermal profiles of DPP-TPA NPs in PBS with different concentrations under laser irradiation (660 nm, 1 W/cm2). (f) Photothermal profiles of DPPTPA NPs in a mixture of PBS and THF under laser irradiation (660 nm, 1 W/cm2).

TPA NPs (Figure S1). The UV−vis absorption spectrum shows that DPP-TPA has good NIR absorption with the maximum peak at 630 nm in dichloromethane due to its extended conjugated structure. With the formation of DPP-TPA NPs, the absorption spectrum displays an obvious bathochromic-shift with the maximum absorption peaks at 660 nm in PBS (Figure 1b) that can be ascribed to the increased interchain interactions induced by self-assemble, which is more suitable for both imaging and therapy in vivo. The morphology and particle size have been studied by transmission electron microscope (TEM) and dynamic light scattering (DLS). TEM image shows that DPP-TPA NPs exhibit spherical morphology with a diameter less than 100 nm (Figure 1c), which can provide the NPs tumor targeting property based on the EPR effect. DLS analysis further indicates the DPP-TPA NPs present uniform size dispersion in PBS solution with an average size of ∼76 nm (Figure 1d). To confirm the multifunction of DPP-TPA NPs, its photoacoustic (PA) property, photothermal conversion efficiency, and reactive oxygen species generation are examined in PBS solution, respectively. As shown in Figure 2a, the solution of DPP-TPA NPs presents an obvious PA signal. And the PA intensity enhances obviously along with the concentration increasing. When the concentration of DPP-TPA NPs reaches 80 μg/mL, the PA image presents the brightest signal. And the relationship between the concentration of DPP-TPA NPs and PA intensity is linear with R2 = 0.98276 (Figure 2b). The singlet oxygen (1O2) generation ability of DPP-TPA NPs is measured by monitoring the photooxidation of 1,3-diphenylisobenzofuran (DPBF) at 418 nm irradiated by 660 nm laser (Figure S2). As shown in Figure 2c, the absorption intensity at 418 nm of the DPP-TPA and DPBF mixture decreases significantly with laser power increase. Especially, at the

guided PTT/PDT performance and photothermal conversion mechanism. Herein, a single component DPP-based organic nanoagent is synthesized for PAI-guided PTT/PDT. Triphenylamine (TPA) as a typical donor53 is conjugated with DPP core to form a NIR-absorbed D-A-D molecule 3,6-bis(5-(4-(diphenylamino)phenyl)thiophen-2-yl)−2,5-bis(2-ethylheptyl)pyrrolo[3,4 -c] pyrrole-1,4(2H,5H)-dione (DPP-TPA). The photon-induced charge carrier dynamics in DPP-TPA is examined by steadystate and time-resolved photoluminescence, which verifies that additional nonradiative decay channels are opened after conjugation, and the excited-states energy of one segment could be consumed by the attached segment through phonon coupling (generate heat). After reprecipitation,54 the DPP-TPA can self-assemble into NPs and disperse in aqueous solution, which exhibits passive targeting to tumor sites by the enhanced permeability and retention (EPR) effect55 and makes tumor treatment visualized by photoacoustic imaging (Scheme 1). In vitro and in vivo experiments further demonstrate that the single component DPP-TPA NPs with multifunction can generate heat and ROS simultaneously induced by a single laser (660 nm) and greatly inhibit tumor growth by synergistic PTT and PDT under the guidance of PAI.

RESULTS AND DISCUSSION Synthesis and Characterization. Scheme S1 shows the synthetic procedure of the DPP-TPA molecule. TPA is introduced into the DPP core to form a D-A-D structure. Using the reprecipitation method, hydrophobic DPP-TPA could be prepared NPs and uniformly dispersed in phosphate buffer (PBS, pH = 7.4) with cyan color (Figure 1a). After irradiation over a long time with laser, there is almost no photodegradation, revealing excellent photostability of DPP1056

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Figure 3. (a) Absorption spectra of samples dissolved in toluene with a concentration of 10−5 mol/L. The dashed line is a guide to show the excitation position at 325 nm. (b) Photoluminescence spectra of samples dissolved in toluene (10−4 mol/L). The relative intensities are normalized by the absorbance values at 325 nm. (c) Time-resolved photoluminescence decay transients at the specific emission ranges. Solid lines represent fits with single- or biexponential decay functions. The optical excitation is performed with 325 nm pump pulses (50 fs, 1 kHz, ∼1 μJ/cm2).

Figure 4. (a) Fluorescence images of DPP-TPA NPs in HeLa cells excited by 660 nm laser (left panel, fluorescence; middle panel, DAPI; right panel, merged images). (b) Fluorescence images of DPP-TPA NPs and DCFH-DA in HeLa cells excited at 488 nm laser (left panel, DCF fluorescence; middle panel, DAPI; right panel, merged images). (c) Cell viabilities of HeLa cells after treatment with various concentrations of DPP-TPA NPs in different conditions.

power intensity of 1.0 W/cm2, the absorption peak completely disappears under the irradiation in less than 30 s. The calculated yield of singlet oxygen is ∼33.6%, which indicates that DPP-TPA NPs present excellent 1O2 generation ability for PDT. Using 660 nm laser as the excitation light, the photothermal conversion efficiency of DPP-TPA NPs (80 μg/mL in PBS) is investigated systematically. As shown in Figure 2d, with the increase of laser power, the temperature of the solution becomes higher and higher. When the laser power reaches 1 W/cm2, the temperature of the solution rapidly arrives 50 °C within a short time (about 100 s). After that, the temperature slowly increases to over 60 °C, which can effectively ablate tumor cells. The photothermal conversion efficiency of DPPTPA NPs is ∼34.5%, which is higher than many photothermal agents reported in previous literature.56,57 Figure 2e shows the relationship between DPP-TPA NPs concentrations and temperature under laser irradiation (660 nm, 1 W/cm2), indicating that the concentration of NPs has a large effect on the temperature. When the concentration of DPP-TPA NPs comes to 80 μg/mL, the temperature of solution quickly increases from 25 to 43 °C in less than 60 s. Figure 2f shows the photothermal profiles of DPP-TPA NPs in mixed solution of PBS and THF (80 μg/mL) under laser irradiation. It can be found that the increase rate of temperature became slower with the addition of THF (good solvent of DPP-TPA) into the PBS solution. When the volume ratio of THF increases to 100%, the self-assembled DPP-TPA NPs disaggregate totally in the

mixture and the maximum temperature increases slightly (∼10 °C). This result further confirms that the photothermal conversion efficiency of DPP-TPA NPs in PBS solution can be greatly enhanced beyond that of DPP-TPA in organic solution. To explore the light-to-heat conversion mechanism of the DPP-TPA conjugated molecule, the photon-induced charge carrier dynamics in DPP-TPA, DPP, and TPA are examined by steady-state and time-resolved photoluminescence (TRPL).58−61 Except for an absorption red shift, the typical absorption spectra (Figure 3a) indicate that the absorption peaks of DPP-TPA at around 300 and 640 nm are obviously broadened and enhanced over that of DPP and TPA alone. This phenomenon indicates that more light could be absorbed by the conjugated DPP-TPA system than by the individual molecules under the same experimental conditions. However, the radiative recombination quantum yields (QYs) of DPP and TPA segments are greatly reduced after the conjugation to form DPP-TPA. As shown in Figure 3b, the relative photoluminescence QYs are 1, 0.03, and 0.09, respectively, for DPP, TPA, and DPP-TPA. These relative photoluminescence QY measurements are well consistent with the TRPL observations (Figure 3c). Both of DPP and TPA segments photoluminescence lifetimes are greatly decreased after conjugation. For example, the photoluminescence lifetime of DPP is reduced from 6.8 to 2.6 ns and TPA is reduced from 2.2 to 1.2 ns. These results suggest that additional nonradiative decay channels are opened after conjugation. And the excitedstates energy of one segment could be consumed by the 1057

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Figure 5. (a) PA images in tumor sites after intravenous injection of DPP-TPA NPs (0.2 mg/kg, n = 4) in tumor-bearing mice. (b) Corresponding PA intensity at different time in vivo. (c) 3D PA images of tumor sites at 0 and 2 h postinjection of DPP-TPA NPs. (d) Infrared thermal images of tumor sites after injecting PBS and DPP-TPA NPs for 2 h under irradiation different time.

Figure 6. (a) Relationship between tumor volumes and time for various treatment groups. (b) Body weight changes with time for various treatment groups. (c) Photographs of mice after 16 days treatment.

attached segment through phonon coupling (generate heat). Therefore, it can be concluded that the light-to-heat conversion of the DPP-TPA originates from the enhanced light absorption and reduced light emission after the conjugation. Cellular Investigation of DPP-TPA NPs. Figure 4a shows the fluorescence images of DPP-TPA NPs (10 μg/mL, 200 μL)

in HeLa cells after 24 h incubation. Obviously, bright red color fluorescence indicates DPP-TPA NPs are abundantly dispersed in the cytoplasm of the cell. It also suggests that DPP-TPA NPs exhibit good uptake in tumor cells. Using 2′,7′-dichlorofluorescein diacetate (DCFH-DA, itself having no fluorescence, can form fluorescent 2′,7′-dichlorofluorescein (DCF) after reacting 1058

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Figure 7. H&E stained images of major organs (heart, liver, spleen, lung, and kidney) for different groups after 16 days treatment.

with ROS) as an ROS probe,62 the intracellular ROS generation of DPP-TPA NPs are investigated in HeLa cells. As shown in Figure 4b, strong green fluorescence can be observed excited by the laser (488 nm), ascribed to the production of DCF in HeLa cells, which confirms the excellent ROS generation of DPPTPA NPs in tumor cells. Figure 4c shows the cell viability by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay63 in different irradiating conditions. It can be found that the cell viability decreases with the irradiation (660 nm, 1 W/cm2), and the half-maximal inhibitory concentration (IC50) is about 12.5 μg/mL, indicating the efficient PDT/PTT synergetic therapy in tumor cells of DPPTPA NPs. Compared to the irradiation group, the dark group exhibits tiny cell toxicity, indicating the desirable biocompatibility and low toxicity of DPP-TPA NPs to cells. These results confirm the efficient photo toxicity and excellent biocompatibility of DPP-TPA NPs in cells. In Vivo Imaging of DPP-TPA NPs. Figure 5a shows the PA images of a tumor site after intravenous injection of DPP-TPA NPs to mice at different times. After 2 h postinjection, PA signal intensity reaches the maximum at the tumor sites, indicating the excellent PAI ability and tumor targeting of DPPTPA NPs by EPR effect in vivo. Figure 5b indicates the PA signals present a rapid increase from 1 to 4 h after the injectio, and the highest enrichment period is between 2 and 4 h. After 6 h postinjection, the PA signal intensity decreases to the initial level, suggesting DPP-TPA NPs could be catabolized easily in the tumor site. Figure 5c shows the three-dimensional (3D) PA images of the tumor sites at 0 and 2 h postinjection of DPPTPA NPs. The different intensity can be visualized obviously, which also denotes that the PA signals of DPP-TPA NPs are determinable throughout the tumor. According to the PAI results, infrared thermal imaging of DPP-TPA NPs in vivo is further investigated under 660 nm laser after 2 h. As shown in

Figure 5d, the temperature of tumor sites of DPP-TPA NPs injected mice increases rapidly and reaches a plateau of ∼60 °C after 6 min irradiation. However, the temperature of the control group with PBS injection shows no obvious increase, confirming excellent PAI property, photothermal conversion, and the passive tumor targeting performance of DPP-TPA NPs. Imaging-Guided Phototherapy in Vivo. Motivated by the excellent PAI, ROS generation, and photothermal conversion performance of DPP-TPA NPs, the imaging-guided tumor therapy is investigated in vivo. Tumor bearing mice were divided into four groups for various treatments. As shown in Figure 6a, tumor volumes of both 1 W/cm2 laser only group and NPs only group increase rapidly during the entire treatment period. And, the group of NPs injection with lower power (660 nm laser, 0.5 W/cm2) presents a minor inhibiting effect of the tumor growth due to the slower temperature increase and ROS generation. Differently, with the higher power intensity of laser (600 nm, 1 W/cm2), the tumors are totally eliminated in the first treatment. After this treatment, there is no recurrence of the tumors during the entire experimental period, which indicates DPP-TPA NPs has highly synergetic PTT/PDT therapy efficiency and can completely inhibit the tumors growth. The body weights of mice models for every 2 days are recorded in Figure 6b.The growth of mice body weight can be observed for all the groups, indicating the excellent biocompatiability and low toxicity of DPP-TPA NPs in vivo. The representative photographs of mice after different treatments for 16 days are presented in Figure 6c, respectively. The tumor after PDT/PTT treatment completely disappeared, which further demonstrates the excellent synergetic therapy of DPP-TPA NPs in vivo. Ex Vivo Histology Examination. The possible pathomorphology analysis of the major organs (heart, liver, spleen, lung, and kidney) was carried out after 16 days treatment. As shown 1059

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(0.265 g, yield: 48%). 1H NMR (500 MHz, CDCl3) δ 8.90 (d, J = 2.9 Hz, 2H), 7.64 (d, J = 4.4 Hz, 2H), 7.29 (s, 2H), 4.04 (t, J = 7.7 Hz, 4H), 1.88 (s, 2H), 1.47−1.12 (m, 16H)0.89−0.71 (m, 12H). 13C NMR (126 MHz, CDCl3) δ 168.07, 138.42, 135.17, 129.00, 109.43, 77.34, 47.70, 38.73, 30.36, 28.93, 25.00, 17.10, 14.06. Synthesis of DPP-TPA. Under N2 atmosphere, in a twonecked bottle, 2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo [3,4-c]pyrrole-1,4(2H,5H)-dione (0.552 g, 1 mmol), 4-bromo-N,N-diphenylaniline (0.808 g, 2.5 mmol), Pd(OAc)2 (0.006 g, 0.025 mmol), pivalic acid (0.015 g, 0.15 mmol), and anhydrous K2CO3 (0.345 g, 2.5 mmol) are stirred in 5 mL of anhydrous dimethylacetamide at 110 °C for 4 h. The mixture is poured into 100 mL of water and extracted with dichloromethane after cooling down to room temperature. The organic layer is washed with brine and dried with anhydrous magnesium sulfate. The solvent is removed by rotary evaporation, and then the crude product is purified by column chromatography (silica gel, DCM/PE = 6:1, v/v). (0.851 g, 82% yield). 1H NMR (300 MHz, CDCl3) δ 8.86 (s, 2H), 7.53 (d, J = 8.5 Hz, 4H), 7.37 (d, J = 2.8 Hz, 2H), 7.31 (dd, J = 13.4, 5.7 Hz, 8H) 7.20−6.91 (m, 16H), 4.09 (t, J = 11.6 Hz, 4H), 1.96 (s, 2H), 1.32−1.22(m, 16H), 0.88−0.79 (m, 12H). 13C NMR (126 MHz, CDCl3) δ148.57, 147.15, 139.68, 136.98, 129.44, 127.92, 126.94, 125.12, 125.01, 123.68, 122.85, 45.95, 39.25, 28.55, 23.09, 14.03, 10.62. MALDI-TOF MS (m/z): [M]+ calcd for C66H66N2O2S2, 1011.39; found, 1011.26. Preparation of DPP-TPA NPs. A 200 μL (2 mg/mL−1) aliquot of DPP-TPA solution in tetrahydrofuran (THF) is added into 5 mL of water under vigorous stirring at room temperature. After the mixture is stirred for 5 min, THF is removed by air blowing. DPP-TPA NPs in the solution are obtained by centrifugation. The size and morphology of DPPTPA NPs are determined by TEM and DLS. Photostability. DPP-TPA NPs (80 μg/mL) PBS solution is irradiated by laser (660 nm, 1 W/cm2) for 0, 5, 10, 15, and 20 min, respectively. The absorbance of DPP-TPA NPs is measured by UV−vis spectrophotometer. Singlet Oxygen Detection. 1,3-Diphenylisobenzofuran (DPBF) is used to detect the generation of ROS. DPP-TPA (10−5 mol/L) is mixed with DPBF (10−5 mol/L) in dichloromethane in a dark room and the absorption spectra are recorded immediately after irradiation (660 nm, 1 W/cm2). Methylene blue is used as a reference object to calculate the ROS production yield of DPP-TPA. In Vitro Photoacoustic Imaging. DPP-TPA NPs (0, 10, 20, 40, 80 μg/mL, respectively) in PBS are prepared for PAI. DPP-TPA NPs solutions are introduced into eppendorf tubes and put in deionized water at a consistent depth. Photoacoustic images are obtained at 680 nm wavelength by Nexus128 small animal photoacoustic imaging system. In Vitro Photothermal Effect. DPP-TPA NPs (0, 10, 20, 40, 80 μg/mL, respectively) in PBS are put into eppendorf tubes and irradiate by 660 nm laser (0.13, 0.25, 0.5, 1.0 W/cm2, respectively) for 10 min. The change of temperature is record by an infrared camera. Cell Lines. Hela cell line and HCT-116 cell line are provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). They are cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS) under 5% CO2 at 37 °C. Cellular Uptake Study. Hela cells incubated with DPPTPA NPs PBS (40 μg/mL, 2 mL) in a confocal dish in a 5%

in Figure 7, the hematoxylin and eosin (H&E) stained slices indicate that the treatment causes no noticeable tissue damage or adverse effect in major organs. On the contrary, compared to the laser-only and NPs-only treatment groups (Figure S3), obvious apoptosis (nucleus become broken, smaller and less) can be observed for the tumor treated by NPs + 0.5 W/cm2 laser group (tumor treated by NPs + 1 W/cm2 laser disappeared completely). These results further indicate that DPP-TPA NPs are not toxic to normal organs and efficiently destroy tumor ability. Therefore, these ex vivo measurements demonstrate that DPP-TPA NPs can serve as an efficient therapeutic agent for PAI-guided tumor PDT/PTT without any appreciable side effect.

CONCLUSIONS In summary, a D-A-D structured organic therapeutic agent (DPP-TPA NPs) with broad NIR absorption has been successfully synthesized for photoacoustic/thermal imaging guided photodynamic/photothermal synergistic tumor therapy. Photon-induced charge carrier dynamics of DPP-TPA is measured by steady-state and time-resolved photoluminescence, indicating that enhanced light absorption and reduced light emission make it present high photothermal conversion efficiency (η = 34.5%). Also, the self-assembly enhanced D-A-D makes DPP-TPA NPs exhibit strong photoacoustic signals and excellent ROS generation (ΦΔ = 33.6%) under laser irradiation. With targeting accumulation in tumor sites by the EPR effect, DPP-TPA NPs present excellent tumor destruction ability through photodynamic and photothermal synergetic therapy with a single wavelength laser (660 nm, 1W/cm2). The design illuminates the photothermal conversion mechanism and provides a D-A-D structured organic therapeutic agent for imaging guided tumor PDT/PTT with enhanced efficiency. MATERIALS AND METHODS Materials and Apparatus. Palladium acetate, 3,6-dithiophen-2-yl-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, pivalic acid, and 4-dimethylaminobenzophenone are purchased from Sigma-Aldrich. Anhydrous dimethylacetamide, 4-bromo-N,Ndiphenylaniline, 3-(bromomethyl)heptane, and N-bromosuccinimide are purchased from Adamas. The 1H NMR and 13C NMR spectra are recorded in CDCl3 with tetramethylsilane as internal standard on a Bruker DRX NMR spectrometer (500 MHz). The absorption spectra are measured on an UV-3600 UV−vis spectrophotometer (Shimadzu, Japan). The size of nanoparticles is recorded by a 90 Plus particle size analyzer (Brookhaven Instruments, USA). The morphology of nanoparticles is carried out on a JEOL JEM-2100 transmission electron microscope (TEM). Photoacoustic images are obtained with a Nexus128 small animal photoacoustic imaging system (Ann Arbor, MI, USA). Thermal images are obtained by an E50 infrared camera (FLIR, Arlington, VA). Synthesis of DPP. Under N2 atmosphere, 3,6-dithiophen-2yl-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione (0.300 g, 1.0 mmol), anhydrous K2CO3 (0.166 g, 1.2 mmol), and 3(bromomethyl)heptane (0.494 g, 2.4 mmol) are added into 15 mL N,N-dimethylformamide. The mixture is stirred at 125 °C for 24 h, then poured into 150 mL water and extracted with dichloromethane. The organic layer is washed with brine and dried with anhydrous sodium sulfate. The solvent is removed by rotary evaporation, and the crude product is purified by column chromatography (silica gel, DCM/PE = 1:2, v/v). 1060

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ACS Nano CO2 incubator at 37 °C for 24 h. The medium is removed and rinsed with PBS. The images are viewed with Olympus IX 70 inverted microscope, and the samples are excited at 660 nm laser and collect from 660 to 700 nm. The cells are stained with DAPI (nuclei-specific dye). Cytotoxicity Assay. DPP-TPA NPs PBS is diluted to various concentrations by DMEM. Hela cells are divided into three groups in 96-well cell-culture plates. DPP-TPA NPs are added in the plates with same volume (200 μL) in control wells. The first group of cells is maintained in darkness, the second group is irradiated by laser (660 nm, 0.5 W/cm2, 5 min) after 24 h, and the last group is irradiated by laser (660 nm, 1 W/cm2, 5 min). The cells are incubated for 48 h more and incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (500 μg/mL) at 37 °C for 4 h in 5% CO2, then treated with 150 μL of DMSO. The absorption is measured by Bio-Tek microplate reader at 570 nm. Fluorescence Images of Cellular ROS. Hela cells are incubated with DPP-TPA NPs (40 μg/mL, 2 mL) in a confocal dish in darkness for 24 h, then the cells are incubated with 2,7dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM, 0.2 mL) for another 1 h (660 nm laser, 1 W/cm2, 5 min). The fluorescence images are obtained by an Olympus IX 70 inverted microscope, and the samples are excited at 488 nm and collected from 505 to 550 nm. The cells are stained with DAPI (nuclei-specific dye). Animal Models. All animal experiments are performed in compliance with the relevant laws and institutional guidelines, and the institutional ethics committee has approved the experiments. HCT-116 cells are injected into the left flank subcutaneously in athymic nude mice of ∼6 weeks old. These mice are used for photoacoustic/thermal imaging and PDT/ PTT treatment when the volumes of the tumors are ∼100 mm3. In Vivo Photoacoustic and Thermal Imaging. A 100 μL aliquot of DPP-TPA NPs (40 μg/mL) PBS is injected through the tail vein into tumor-bearing mice. After that, photoacoustic imaging of tumor sites at different period (0, 1, 2, 4, 6, and 8 h) are performed on a Nexus128 small animal photoacoustic imaging system with 680 nm laser. Thermal imaging is monitored by an E50 infrared camera when the tumor sites are administrated with 660 nm laser (1 W/cm2) for different times (0, 2, 4, 6, 8 min) after 2 h postinjection of DPP-TPA NPs. In Vivo PDT/PTT. HCT-116 tumor-bearing nude mice are randomly divided into four groups with the same number of male and female (PBS with laser treatment, DPP-TPA NPs only, DPP-TPA NPs with 0.5 W/cm2 laser, DPP-TPA NPs with 1 W/cm2 laser). For each group, mice are injected with 100 μL of DPP-TPA NPs (40 μg/mL) PBS or PBS through the tail vein, respectively. Two hours later, mice are irradiated with laser (660 nm, 1 W/cm2 or 660 nm, 0.5 W/cm2) for 8 min. The treatments are conducted every other day. Tumor volumes and body weights of mice are record every other day as well. Tumor volumes are calculated by the commonly used equation: V= width2 × length/2. Ex Vivo Histology Examination. After treatment, tumorbearing mice are sacrificed and tumor, heart, liver, spleen, lung, and kidney are taken out for histology analysis. After dehydration and staining with hematoxylin and eosin (H&E), these tissues are embedded in paraffin cassettes. The H&E stained images are viewed by a microscope.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b07927. Synthetic route of DPP-TPA (Scheme S1); UV−vis absorption spectra of DPP-TPA NPs in PBS (pH = 7.4) under irradiation for different time (Figure S1); absorption intensity of DPP-TPA (10−5 mol/L) mixed with DPBF (2 × 10−5 mol/L) in DCM with the power density (660 nm laser) of 0.25, 0.5, 0.75, and 1 W/cm2, respectively, over time (Figure S2); H&E stained images of tumors from control groups (Figure S3) (PDF)

AUTHOR INFORMATION Corresponding Authors

* E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Wei Huang: 0000-0001-7004-6408 Xiaochen Dong: 0000-0003-4837-9059 Notes

The authors declare no competing financial interest.

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