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Diketopyrrolopyrrole-Based Photosensitizers Conjugated with Chemotherapeutic Agents for Multimodal Tumor Therapy Yu Cai, Pingping Liang, Qianyun Tang, Weili Si, Peng Chen, Qi Zhang, and Xiaochen Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09025 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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ACS Applied Materials & Interfaces

Diketopyrrolopyrrole-Based Photosensitizers Conjugated with Chemotherapeutic Agents for Multimodal Tumor Therapy §

‡

Yu Cai,† Pingping Liang,† Qianyun Tang,† Weili Si,† Peng Chen,*, Qi Zhang,*, Xiaochen Dong*,† †

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China. ‡

School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South Puzhu

Road, Nanjing 211816, China. §

School of Chemical and Biomedical Engineering, Nanyang Technological University, 62

Nanyang Drive, 637459, Singapore. Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. KEYWORDS: multifunctional agents; diketopyrrolopyrrole; chemotherapy; photodynamic therapy; photothermal therapy

ABSTRACT:

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For synergistic cancer therapy, it is highly desirable to devise a single multifunctional agent to combine photodynamic therapy (PDT), photothermal therapy (PTT) and chemotherapy, which is soluble and excitable at low irradiation, as well as able to selectively target tumors and achieve high efficacy. Towards this ambition, here, the chemotherapy drugs, chlorambucil (Cb) and all trans retinoic acid (ATRA), are covalently conjugated onto a small dye molecule diketopyrrolopyrrole (DPP-Cb and DPP-ATRA). The soluble nanoparticles (NPs) of DPP-Cb and DPP-ATRA formed by reprecipitation can selectively accumulate in tumors, release chemotherapy drugs under acidic conditions, and exhibit efficient reactive oxygen species (ROS) generation and photothermal conversion under the irradiation of a low power xenon lamp (40 mW/cm2). We show in vitro and in vivo that both NPs can effectively kill cancer cells and suppress cancer growth at a low dose (0.4 mg/kg).

1. INTRODUCTION Chemotherapy is currently the primary clinical choice to treat malignant tumors. But the chemotherapy agents usually suffer from poor water solubility, non-specificity, low bioavailability, rapid blood/renal clearance, low accumulation in tumors, severe multidrug resistance (MDR), and adverse off-target side effects.1-5 To tackle these problems, drug carriers including liposomes vesicles,6 inorganic materials,7 polymeric nanoparticles8-9 have been developed to encapsulate the chemotherapy agents. But many carriers have low drug loading capacity and cause biotoxicity.10-12 In recent years, phototherapy has attracted enormous interests for cancer treatment, because of the minimal invasiveness and low systemic damage.13-14 Phototherapy includes photodynamic


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therapy15-16 (PDT, relying on photosensitizers to generate reactive oxygen species for killing tumor cells) and phototheramal therapy17-18 (PTT, relying on photothermal agent to convert light energy into heat for tumor ablation). However, the efficacy of the former is plagued by the low oxygen level in tumors while the latter requires high laser power which leads to lateral tissue damage. Therefore, various organic photosensitizers (e.g., indocyanine green, phthalocyanine and chlorin e6) have been incorporated with inorganic photothermal agents (e.g., nanographene oxide, gold nanostructures, and silica nanoparticles) for dual phototherapy (PDT and PTT) to achieve synergistic enhancement.19-23 Nevertheless, incorporation of two distinct functional components demands complex preparation and two excitation wavelengths (hence prolonged and complicated treatment). It is highly desirable to devise a single multifunctional agent to combine PDT, PTT and chemotherapy, which is soluble and excitable at low irradiation, as well as able to selectively target tumors and achieve high efficacy. A few attempts have been made towards this ambition. Li et al. integrated three distinct functional components including a polymeric matrix as the carrier, gadolinium(III) chelated chlorin e6 as the PDT photosensitizer, and a pluronic prodrug (doxorubicin).24 Under strong laser illumination, such micelles exhibit certain PTT effect. But synthesis of such multi-component system is complicated and tedious. In the work of Zhang et al., hollow mesoporous CuS nanoparticles serve simultaneously as the drug carrier for doxorubicin, PTT agent, and PDT photosensitizer (but with unclear mechanism).25 But the longterm toxicity of inorganic nanomaterials is of a serious concern. Diketopyrrolopyrrole (DPP) is a small dye molecule with good photo-stability, good absorption of light (ensuring good tissue penetration depth), and ease to be functionalized, which can serve as both effective photothermal agent and photosensitizer. Here, the chemotherapy drugs,


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chlorambucil26 (Cb) and all trans retinoic acid27 (ATRA), are covalently conjugated onto DPP (DPP-Cb and DPP-ATRA). The soluble nanoparticles (NPs) of DPP-Cb and DPP-ATRA formed by reprecipitation28 can selectively accumulate in tumors via enhanced permeability and retention (EPR) effect, and exhibit efficient reactive oxygen species (ROS) generation and photothermal conversion under the irradiation of a low power xenon lamp (> 40 mW/cm2). In addition, Cb and ATRA molecules are released from the NPs due to breaking of the ester bond as triggered by the weakly acidic microenvironment in tumor (pH 6.0-6.8) and acidic condition in lysosomes (pH ~5.0).29 Because of the synergist effects from PDT, PTT and chemotherapy (Scheme 1), we show in vitro and in vivo that both NPs can effectively kill cancer cells and suppress cancer growth at a low dose (0.4 mg/kg). 2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization The synthetic route is outlined in Scheme 2 (as detailed in experimental section). DPP, DPPATRA and DPP-Cb are successfully prepared with high yield. NPs of DPP-ATRA and DPP-Cb formed by reprecipitation can be well dispersed in phosphate buffer (PBS) presenting crimson color (Figure S1). UV-Vis absorption spectra of DPP, DPP-ATRA and DPP-Cb NPs in PBS solution all exhibit the characteristic peak of DPP at ~560 nm (Figure 1a). And the successful conjugation of ATRA and Cb onto DPP is confirmed by their characteristic peak at ~360 and ~258 nm respectively. As revealed by scanning electron microscopy (SEM), DPP-ATRA and DPP-Cb NPs show cubic morphology with the dimension below 100 nm (Figure 1b and 1c), and the morphology of the NPs relates to the stirring time and speed in the dispersions. The dynamic light scattering (DLS) analysis further revealed that the average size of DPP-ATRA NPs was ~70


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nm and DPP-Cb NPs was ~60 nm (Figure S2). Such size falls into the optimal range of 30-200 nm to make them suitable for passive tumor targeting because of EPR effect.30 2.2. Multi-Functionalities in Vitro To confirm the multi-functionalities of the conjugations, the singlet oxygen (1O2) generation, photothermal conversion efficiency and cumulative release of Cb and ATRA are firstly examined in solutions, respectively. The detection of 1O2 generation is by using reported 1, 3diphenylisobenzofuran (DPBF), whose absorption peak at 418 nm will be weakened upon reaction with 1O2.31 As shown in Figure 2, the absorption intensity of DPBF decreases drastically with the increasing irradiation duration by a Xenon lamp (> 510 nm, 40 mW/cm2), indicating the excellent 1O2 generation ability of DPP-Cb (Figure 2a) and DPP-ATRA (Figure 2b). The same Xenon lamp irradiation is also used to stimulate the photothermal conversion. The temperatures of both NP solutions rapidly increase from 25 °C to 40 °C within 2 min and reach ~45 °C in 10 min (Figure 2c and Figure S3), which is sufficient to ablate tumor cells. The in vitro release behavior of DPP-Cb and DPP-ATRA NPs in PBS is evaluated by dialysis in PBS at 37 °C. It is evident that the release of Cb or ATRA from the NPs is promoted by low pH (Figure 2d), confirming that weakly acidic environment in tumor tissue (pH 6.5) and acidic environment in intracellular lysosomes (pH 5.0) can trigger the drug release. 2.3. Cellular Uptake and Cytotoxicity DPP-ATRA and DPP-Cb NPs fluoresce with the emission peak at ~620 nm (Figure S4) in PBS, suggesting that the cellular uptake of these NPs can be conveniently visualized. As revealed by confocal fluorescence imaging, both types of NPs can be readily uptaken into cytosol (but not nucleus) by HeLa cells after 24 h incubation (Figure S5). In addition, co-localization imaging


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shows that many DPP-Cb or DPP-ATRA NPs are segregated into the lysosomes (Figure 3), within which drug release is stimulated. Furthermore, we use 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) as the probe to detect the intracellular generation of reactive oxygen species (ROS) which cleaves no-fluorescent DCFHDA to fluorescent 2’, 7’-dichlorofluorescein (DCF).32 As shown in Figure 4a and b, after irradiation (> 510 nm, 40 mW/cm2) of the HeLa cells incubated with DPP-Cb or DPP-ATRA NPs together with DCFH-DA, strong green fluorescence can be observed at 488 nm excitation. This observation confirms the ability of DPP-Cb and DPP-ATRA NPs for light-triggered production of ROS inside tumor cells. The cytotoxicity in vitro was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa cells.33 As demonstrated in Figure 4c, the cytotoxicity of both free Cb and ATRA molecules does not reach the half maximal inhibitory concentration (IC50) even at a rather high concentration of 60 µM. And free DPP molecules present a low cytotoxic effect in dark but high phototoxicity (IC50 ~55 µM), suggesting its suitability for phototherapy. Subsequently, cytotoxicity of DPP-Cb and DPP-ATRA NPs to HeLa cells is measured with treatment. In addition, we show that both DPP-Cb and DPP-ATRA NPs exhibit good biocompatibility in dark and high phototoxicity (Figure 4d and e). Moreover, as compared to the individual free molecules, NPs can achieve much lower effective dose testifying the synergistic effects enabled by such multi-functional NPs. Specifically, after culturing for 48 h and irradiation for 10 min, the IC50 value of the NPs is only ~14 nM. 2.4. Photothermal Conversion


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Taking the advantage of the photothermal conversion ability of the DPP-Cb and DPP-ATRA NPs, the infrared thermal imaging is employed for real-time in vivo monitoring the tumor targeting property of these NPs. Figure 5a shows the infrared thermal images of the tumor sites after intravenous injection of PBS, DPP-Cb NPs or DPP-ATRA NPs (80 µg/mL) to HeLa tumorbearing mice at different times, under the illumination with a xenon lamp (> 510 nm, 40 mW/cm2) for 10 minutes every 2 hours. In contrast to PBS control, the tumor temperature (but not the body temperature) increases to ~45 °C 4h after the injection of DPP-Cb or DPP-ATRA NPs (Figure 5b). This observation means that both NPs can selectively target tumor tissues and their photothermal effect is sufficient to ablate the tumor. It is noted that the tumor temperature returns to normal after 8h, meaning that both NPs are readily metabolized (Figure 5b). 2.5. Multimodal Tumor Therapy in Vivo To examine the therapeutic effectiveness of our multi-functional NPs in vivo, 20 HeLa tumorbearing mice were randomly divided into 5 groups for different treatments. As presented in Figure 6a and b, the tumor volumes of the control group increase quickly over the observation period (24 days). The mice injected with DPP-Cb or DPP-ATRA NPs (80 µg/mL, 100 µL) without illumination show apparent suppression in tumor growth, attributable to the anticancer drugs being released from the NPs. Remarkably, DPP-Cb or DPP-ATRA NPs injection followed by xenon lamp irradiation (> 510 nm, 40 mW/cm2) can greatly inhibit the tumor growth. As shown in Figure 6c, all the mouse groups gain body weight similarly, indicating the good biocompatibility of the NPs. 2.6. Ex Vivo Histology Examination Figure 6d shows the tumor histologic section of each group using hematoxylin and eosin (H&E) staining.34 The cells in the control group exhibit spindle shape and large nucleus, consistent with


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the observed rapid tumor growth. Distinctly, for the DPP-Cb or DPP-ATRA NP treated groups the cellularity of the tumor tissue significantly decreases, and nuclear fragmentation and shrinkage are observed. For the group combing NP treatment and illumination, large necrotic areas can be observed. H&E staining of the major organs (heart, liver, spleen, lung, kidney) from each group is also analysed and shown in Figure 7. There is no obvious difference between these groups, demonstrating no tissue toxicity of DPP-Cb and DPP-ATRA NPs.

CONCLUSIONS In summary, the nanoparticles formed by diketopyrrolopyrrole (DPP) conjugated with chemotherapeutic compounds have been synthesized for multimodal tumor therapy. DPP plays multiple functions, serving as the sensitizer for photodynamic therapy, the agent for photothermal therapy, as well as the drug carrier. Two chemotherapeutic compounds (Cb and ATRA) have been examined, indicating the universality of our strategy. The desirable size of the nanoparticles ensures their good solubility, ability to selectively accumulate in tumors via EPR effect, as well as the ease to be metabolized after treatment. We demonstrate that due to the synergistic effect the NPs are able to achieve much lower effective dose, promising for clinical applications. 3. MATERIALS AND METHODS 3.1. Chemicals and Characterizations 3,6-Di-thiophen-2-yl-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, Chlorambucil (Cb), all trans retinoic acid (ATRA), 4', 6-diamidino-2-phenylindole (DAPI), 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) and 1, 3-diphenylisobenzofuran (DPBF) were purchased from SigmaAldrich (MO, USA). Hexamethylene dibromide, potassium hydroxide, N, N-dimethylformamide


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(DMF) were purchased from Adamas (China). Lyso-Tracker Green was purchased from Molecular Probes® (USA). Other reagents or solvents were used as received without further purified of analytical grade. The 1H NMR and 13C NMR spectra were measured on Bruker DRX NMR spectrometer in CDCl3 and tetramethylsilane (TMS) was selected as the internal standard. Absorption spectra were measured on an UV-3600 UV-Vis-NIR spectrophotometer (Shimadzu, Japan). Fluorescence emission spectra were recorded on an F-7000 spectrometer (HITACHI, Japan). The morphology of nanoparticles was photographed on a scanning electron microscope (SEM, Hitachi S-4800, Japan). Thermal images were photographed by an E50 infrared camera (FLIR, Arlington, VA). The cell imaging was viewed by an Olympus IX 70 inverted microscope. All experiments were performed in compliance with the relevant laws and institutional guidelines, and the institutional committee(s) has approved the experiments. 3.2. Synthesis of DPP 3,6-di-thiophen-2-yl-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

(1.24

g,

5.0

mmol),

hexamethylene dibromide (6.02 g, 12 mmol) and potassium hydroxide (0.67 g, 12 mmol) were added into 20 mL N,N-dimethylformamide under N2 atmosphere. The mixture was stirred overnight at 25 oC then poured into 100 mL water, extracted with chloroform and washed with water and brine, then dried with anhydrous sodium sulfate. The solvent was removed using rotary evaporation and purified by column chromatography (silica gel, PE/DCM=3:1, v/v). (1.62 g, yield: 52%). 1H NMR (400 MHz, CDCl3) δ 8.91 (dd, J = 3.9, 1.1 Hz, 2H), 7.62 (dd, J = 5.0, 1.1 Hz, 2H), 7.25 (dt, J = 8.9, 4.4 Hz, 2H), 4.09–3.99 (m, 4H), 3.39 (dt, J = 13.6, 6.8 Hz, 4H), 1.89–1.65 (m, 16H). 13C NMR (101 MHz, CDCl3) δ 161.24, 139.89, 135.04, 130.10, 128.51, 107.61, 77.19, 41.83, 33.70, 33.07, 31.69, 29.75, 28.48, 25.83, 25.48, 24.53.


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3.3. Synthesis of DPP-ATRA 2,5-Bis-(6-bromo-hexyl)-3,6-di-thiophen-2-yl-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione (3.14g, 5 mmol), potassium carbonate (1.38 g, 10 mmol) and all-trans retinoic acid (3.6 g, 12 mmol) were dissolved in 20 mL of N,N-dimethylformamide under N2 atmosphere. The reaction mixture was stirred at 30 °C for 12 h. After that, the mixture was poured into 100 mL water, extracted with chloroform and washed with water and brine, then dried with anhydrous sodium sulfate. The solvent was removed using rotary evaporation and purified by column chromatography (silica gel, PE/DCM=1:1, v/v). (3.73 g, yield: 72%). 1H NMR (400 MHz, CDCl3) δ 8.91 (d, J = 3.9 Hz, 2H), 7.64 (d, J = 5.0 Hz, 2H), 7.29 (d, J = 4.8 Hz, 2H), 6.98 (dd, J = 14.9, 11.3 Hz, 2H), 6.27 (d, J = 15.3 Hz, 4H), 6.14 (s, 2H), 6.11 (s, 2H), 5.74 (d, J = 10.1 Hz, 2H), 4.11 (m, 4H), 3.67 (m, 4H), 2.49 (m, 6H), 2.32 (m, 6H), 2.01 (m, 4H), 1.86–1.66 (m, 22H), 1.52–1.19 (m, 20H). 13C NMR (126 MHz, CDCl3) δ 173.13, 167.14, 161.26, 151.53, 139.41, 137.73, 137.31, 135.79, 135.27, 132.32, 130.24, 129.88, 129.80, 129.09, 128.22, 125.48, 118.59, 118.51, 117.37, , 63.86, 42.42, 39.67, 34.51, 32.60, 31.37, 30.33 (s), 29.26, 28.96, 27.76, 26.01, 25.70, 21.18, 20.15, 19.26, 13.97, 12.77. MALDI-TOF MS (m/z): [M]+ calcd for C66H84Cl4N2O6S2, 1065.51; found, 1065.9342. 3.4. Synthesis of DPP-Cb 2,5-Bis-(6-bromo-hexyl)-3,6-di-thiophen-2-yl-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione (3.14g, 5 mmol), potassium carbonate (1.38 g, 10 mmol) and chlorambucil (3.6 g, 12 mmol) were dissolved in 20 mL of N,N-dimethylformamide under N2 atmosphere. The reaction mixture was stirred at 30 °C for 12 h. After that, the mixture was poured into 100 mL water, extracted with chloroform and washed with water and brine, then dried with anhydrous sodium sulfate.


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The solvent was removed using rotary evaporation and purified by column chromatography (silica gel, PE/DCM=1:1, v/v). (2.78 g, yield: 69%). 1H NMR (300 MHz, CDCl3) δ 8.92 (d, J = 3.8 Hz, 2H), 7.63 (d, J = 4.9 Hz, 2H), 7.29 (d, J = 4.5 Hz, 2H), 7.06 (d, J = 8.4 Hz, 4H), 6.61 (d, J = 8.5 Hz, 4H), 4.07 (dd, J = 13.5, 6.9 Hz, 4H), 3.68 (d, J = 6.1 Hz, 4H), 3.62 (d, J = 6.2 Hz, 16H). 2.55 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.96–1.58 (m, 20H). 13C NMR (126 MHz, CDCl3) δ 173.17, 160.97, 144.21, 141.79, 139.60, 135.71, 130.61, 129.56, 129.46, 127.39, 122.24, 112.12, 67.69, 53.48, 44.26, 42.53, 35.98, 34.76, 31.51, 28.47, 27.89, 27.03, 26.62. MALDI-TOF MS (m/z): [M]+ calcd for C54H66Cl4N4O6S2, 1073.07; found, 1072.9286. 3.5. Preparation of DPP, DPP-ATRA and DPP-Cb NPs 200 µL of 2 mg/mL–1 of DPP, DPP-ATRA or DPP-Cb in tetrahydrofuran solution was added into 10 mL water with vigorous stirring at room temperature, respectively. After that, the mixture was stirring for 5 min, then, tetrahydrofuran was removed by nitrogen-blowing. At last, the NPs in the solution were achieved by centrifugation. The morphology and size were determined by SEM, respectively. 3.6. Singlet Oxygen Detection 1,3-diphenylisobenzofuran (DPBF) was used to detect the singlet oxygen generation of DPPATRA and DPP-Cb. DPP-ATRA or DPP-Cb (10-5 mol/L) was mixed with DPBF (6*10-5 mol/L) in dichloromethane solution at a dark room, and the absorption spectra of the mixture were immediately measured after irradiation with xenon lamp (> 510 nm, 40 mW/cm2) over time. Methylene blue as a reference was used to calculate the 1O2 production yield of DPP-ATRA and DPP-Cb. 3.7. In Vitro Photothermal Effect


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DPP-Cb and DPP-ATRA NPs in PBS (40 µg/mL) were respectively introduced into eppendorf tubes and irradiated with xenon lamp (> 510 nm, 40 mW/cm2) for 10 min. The temperature was recorded by an E50 infrared camera. 3.8. In Vitro Drug Release 2 mL of DPP-Cb or DPP-ATRA NPs (0.5 mg/mL) was transferred into a dialysis tube (MWCO = 1000), respectively. They were incubated at 37 °C in 60 mL PBS (pH 5.0, 6.5, 7.4) at predetermined time intervals. Then 2 mL of ambient buffer solution was collected and replaced with 2 mL of fresh PBS (pH 5.0, 6.5, 7.4). The amount of released Cb or ATRA was determined by using an UV-3600 UV-Vis-NIR spectrophotometer (Shimadzu, Japan). 3.9. Cell Line HeLa cell line was provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China), which was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under 5% CO2 and 95% air atmosphere at 37 oC. The cells were subcultured 2-3 times per week by treating with 0.25% trypsin–ethylene diamine tetraacetic acid (EDTA) solution. 3.10. Cellular Uptake Study HeLa cells incubated with DPP-Cb or DPP-ATRA NPs in a confocal dish (40 µg/mL, 2 mL) at 37 oC in a humidified 5% CO2 atmosphere for 24 h, respectively. After that, the cells were washed with PBS solution. The experiment repeated for 4 times and the imaging was presented by Olympus IX 70 inverted microscope, samples were excited at the wavelength of 540 nm and collected from 580 to 640 nm. The cells were stained with nuclei-specific dye, DAPI.


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3.11. Subcellular Location Study HeLa cells incubated with DPP-Cb or DPP-ATRA NPs in a confocal dish (40 µg/mL, 2 mL) at 37 oC in a humidified 5% CO2 atmosphere for 24 h, respectively. Then the cell culture was removed, adding Lyso-Tracker Green dyeing liquid (100 nM) and incubated for another 30 min. After that, the medium was removed and washed with PBS solution and HeLa cells were fixed for 20 min with formalin. The experiment repeated for 4 times and the imaging was presented by Olympus IX 70 inverted microscope, samples were excited at the wavelength of 480 nm and gathered from 500 to 520 nm. 3.12. Fluorescence Images of Cellular ROS In darkness, HeLa cells respectively incubated with DPP-Cb or DPP-ATRA NPs (40 µg/mL, 2 mL) in 2 confocal dishes in a humidified 5% CO2 atmosphere for 24 h at 37 oC. Then the cells were incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 µM) for 1 h and irradiated by xenon lamp (> 510 nm, 40 mW/cm2) for 4 min. The experiments were repeated for 4 times, and the ROS fluorescence imaging was observed by Olympus IX 70 inverted microscope with the excitation at 488 nm and fluorescence emission from 505 to 550 nm. The cells were stained with nuclei-specific dye, DAPI. 3.13. Cytotoxicity Assay Free Cb, ATRA, DPP, DPP-Cb or DPP-ATRA NPs were diluted to various concentrations by DMEM for usage. HeLa cells were cultured in 96-well cell-culture plates. Free Cb and ATRA were added in the plates with same volume (200 µL) in control wells. Double group of DPP was also added in the plates with 200 µL volume ,one group was in dark and the other group was irradiated by xenon lamp (> 510 nm, 40 mW/cm2, 4 min) after 24 h. DPP-Cb or DPP-ATRA NPs


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were measured in the same way with irradiation or not. The cells were incubated for another 48 h, then incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (500 µg/mL) in 5% CO2 at 37 °C for 4 h, and treated with 150 µL of DMSO. The absorption at 570 nm was measured by Bio-Tek microplate reader. 3.14. In Vivo Photothermal Imaging 100 µL of DPP-Cb or DPP-ATRA NPs (40 µg/mL) PBS solution was tail vein injected into HeLa tumor-bearing mice, respectively. Photothermal imaging of tumors was monitored at different time (0, 2, 4, 6 and 8 hours) after irradiation by xenon lamp (> 510 nm, 40 mW/cm2) for 10 min by an infrared camera. 3.15. In Vivo Tumor Treatment 20 nude mice were injected HeLa cells into the armpit as the vivo models. When the volumes of tumor reached ~100 mm3, the mice were randomly divided into 5 groups. Group 1 and 2 were tail vein injected with DPP-Cb or DPP-ATRA NPs PBS solution (40 µg/mL, 100 µL) every 2 days, respectively. Group 3 and 4 were also tail vein injected with DPP-Cb or DPP-ATRA NPs PBS solution (40 µg/mL, 100 µL) every 2 days but irradiated by xenon lamp (> 510 nm, 40 mW/cm2, 10 min) for after injection in 4 h, respectively. Group 5 was injected with saline only. The process above was carried out for 24 days and the tumor size and body weight of the nude mice were measured every other day. 3.16. Histology Examination The nude mice were sacrificed after the treatments for 24 days, and the histology analysis was then conducted. The major organs including hearts, livers, lungs, spleens, kidneys as well as


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tumors from the mice were separated and fixed with 4% formaldehyde solution. After dehydration, all the tissues were embedded in the paraffin cassettes then stained with hematoxylin and eosin (H&E). After that, the imaging was observed by a microscope.

AUTHOR INFORMATION Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The work was supported by the NNSF of China (61525402), Key University Science Research Project of Jiangsu Province (15KJA430006), Six Talent Peaks Project in Jiangsu Province (51235018).

ASSOCIATED CONTENT Supporting Information Supporting Information Available: Photograph of DPP, DPP-Cb and DPP-ATRA NPs (Figure S1), Thermal images of DPP-Cb NPs, DPP-ATRA NPs and PBS before and after illumination (510 nm filter, 40 mW/cm2, 10 min). (Figure S2), Fluorescence emission spectra of DPP, DPPCb and DPP-ATRA NPs in PBS with the excitation at 500 nm. (Figure S3), Fluorescence images of DPP-Cb and DPP-ATRA NPs in HeLa cells. (Scale bar =20 µm) (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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Scheme 1. Schematic diagram of the synergistic PDT/PTT and chemotherapy using DPP-ATRA or DPP-Cb nanoparticles.


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Scheme 2. Synthetic route of DTDPP-ATRA and DPP-Cb. i) N,N-Dimethylformamide, potassium hydroxide, hexamethylene dibromide, overnight, 25 °C, 52%. ii) N,Ndimethylformamide, potassium carbonate, all-trans retinoic acid, 30 °C, 12 h, 72%. iii) N,Ndimethylformamide, potassium carbonate, chlorambucil, 30 °C, 12 h, 69%.


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Figure 1. (a) UV-Vis absorption spectra of DPP, DPP-Cb and DPP-ATRA NPs in PBS (pH=7.4). (b, c) SEM images of DPP-ATRA NPs and DPP-Cb NPs, respectively. The magnified SEM images are shown in insets. Scale bar = 100 nm.


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Figure 2. (a, b) Absorption spectra of 10-5 mol/L DPP-Cb (a) and DPP-ATRA (b) mixed with DPBF (6*10-5 mol/L) under illumination of a Xenon lamp (> 510 nm filter, 40 mW/cm2) with various durations. (c) Photothermal profiles of DPP-Cb and DPP-ATRA NPs in PBS (40 µg/mL) under illumination (Xenon lamp, > 510 nm, 40 mW/cm2). (d) In vitro Cb and ATRA release kinetics from DPP-Cb and DPP-ATRA NPs in PBS under different pH values at 37 °C.


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Figure 3. Fluorescence images of DPP-Cb and DPP-ATRA NPs (red) inside HeLa cells. The lysosomes are labeled by lyso-tracker (green). Scale bar =20 µm.


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Figure 4. (a, b) Fluorescence images the HeLa of incubated with both DCFH-DA and DPP-Cb NPs (a) or DPP-ATRA NPs (b), with excitation at 488 nm. Scale bar =20 µm. (c) In vitro cytotoxicity of free Cb, ATRA, DPP molecules to HeLa cells. *indicates significant differences compared to DPP in dark group (p < 0.05) (d, e) In vitro cytotoxicity of DPP-Cb NPs or DPPATRA NPs to HeLa cells in different conditions. *indicates significant differences compared to 24 h groups in the same darkness or illumination condition (p < 0.05)


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Figure 5. (a) Infrared thermal images after injection of PBS, DPP-Cb and DPP-ATRA NPs of tumor-bearing mice (xenon lamp, > 510 nm, 40 mW/cm2). (b) The body temperature measured at different time. *indicates significant differences of DPP-Cb or DPP-ATRA groups compared to PBS group (p < 0.05)


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Figure 6. (a) Changes of tumor volume for different treatment groups. *indicates significant differences compared to the control group (p < 0.05) (b) Tumors excised ex from the mice after 24 days treatment. (c) Changes of body weight for different treatment groups. (d) H&E stained images of tumors after 24 days treatment of each group of each group.


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Figure 7. H&E staining of heart, liver, spleen, lung and kidney tissues for each mouse group after 24 days treatment.


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