Rational Design of BODIPY-Diketopyrrolopyrrole Conjugated

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Biological and Medical Applications of Materials and Interfaces

Rational Design of BODIPY-Diketopyrrolopyrrole Conjugated Polymers for Photothermal Tumor Ablation Wei Zhang, Wenhai Lin, Chaonan Li, Shi Liu, Xiuli Hu, and Zhigang Xie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10713 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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ACS Applied Materials & Interfaces

Rational Design of BODIPY-Diketopyrrolopyrrole Conjugated Polymers for Photothermal Tumor Ablation Wei Zhang,†,‡ Wenhai Lin,†,‡ Chaonan Li,†,§ Shi Liu,† Xiuli Hu† and Zhigang Xie†,* †Key

Laboratory of Polymer Physics and Chemistry Changchun Institute of Applied Chemistry

Chinese Academy of Sciences Changchun 130022, P. R. China ‡University

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

§University

of Science and Technology of China Hefei 230026, P. R. China

ACS Paragon Plus Environment

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ABSTRACT. Conjugated polymers (CPs) have drawn growing attention in cancer phototherapy and imaging due to their large extinction coefficients, robust photostability, and good biocompatibility. Herein, we propose a new type of photothermal therapy materials on the base of BODIPY-diketopyrrolopyrrole (DPP) CPs, where the number of methyl substitutes at the β and β’ positions on BODIPYs are variable, allowing us to investigate the interplay between the structure of the monomers and the related properties of CPs. Combining the experimental data with theoretical calculation, we concluded that with the decrease of the number of methyl moieties on the β and β’ positions of BODIPY, the polymerization degree and solubility of the obtained CPs were improved, and the polymeric spatial planarization and degrees of conjugation increased, inducing the bathochromic-shift of absorption, which resulted the absorption spectra getting closer to the near-infrared (NIR) region and more conducive to the application of the conjugated polymers in vivo. Afterwards, the CP nanoparticles were made, and their photothermal activity in cancer therapy was validated by a series of in vitro and in vivo experiments. In this paper, we provide a new way to manipulate properties of CPs with great potential in photothermal therapy through the structural engineering.

KEYWORDS: conjugated polymer; BODIPY polymer; diketopyrrolopyrrole; photothermal therapy, theranostic platform


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INTRODUCTION Hyperthermia therapy (PTT), which depends on the capability of photothermal agents (PTAs) to convert absorbed light energy into local hyperthermia to induce deadly cellular damage.1-4 Up to now, a great deal of new materials has been extensively explored in the past few years.5-7 Nevertheless, most of these traditional inorganic or near infrared (NIR) organic dyes still have some disadvantages, such as long retention time in vivo, uncertain biosafety, poor photostability and low light conversion efficiency.8-11 In this respect, conjugated polymers (CPs) are different from other PTAs, which are a kind of π-conjugated macromolecules.12-17 CPs possess several advantages compared with conventional organic dyes and inorganic materials, such as large extinction coefficients, robust photostability, low cytotoxicity, high biocompatibility and versatile functional modification.18-20 Although several CPs have been reported for photothermal therapy, it is still worthy to exploit novel CPs with ideal photothermal activity. Moreover, the interplay of structure and properties of CPs has not been fully understood yet due to the lack of systematic synthesis and comparisons.21,22 Boron dipyrromethene (BODIPY), is a type of excellent organic fluorophore with high absorption coefficients, strong fluorescence quantum yields, and remarkable chemical and photostability.23-28 As far as we know, currently reported BODIPY-based CPs are usually utilized as photoelectric materials, which have rarely been applied in PTT.29-31 Besides, the classic UVvis absorption of BODIPY dyes is mainly located at about 505 nm.32 It is necessary to extend their π-conjugation degree to extend the absorption spectrum to the NIR region.33-35 BODIPYbased CPs provides a simple and efficient tactic to increase the degree of conjugation, and expand the absorption into NIR regions.36-38


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Alteration in the substitution on the β-positions of BODIPY would cause modulations on the dipole of the molecules, which might bring about influences on the band-gap energy and properties of targeted CPs, allowing us to seek out the most favorable CPs materials for biological applications.39,40 Taking the above background into consideration, we synthesized a series of BODIPY-DPP CPs from BODIPY and diketopyrrolopyrrole (DPP) via one-step Suzuki coupling reaction (Scheme 1). With the help of molecular engineering, we hope to explore the impact of different BODIPY structure on the basic properties of the three CPs, such as polymerization degree, solubility and absorption spectra. Furthermore, in order to confirm the applications prospect of BODIPY-DPP CPs in biomedical field, as shown in Scheme 2, polymer P1 and P2 were selected to prepare the corresponding CPNs (P1 NPs and P2 NPs) in the presence of Pluronic F127. The obtained nanoparticles could effectively convert light to heat energy upon irradiation with remarkable photothermal conversion efficiency.

Scheme 1. Illustration of conjugated polymers (P0, P1 and P2) constituted by DPP and BODIPY with different structures.


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RESULTS AND DISCUSSION As outlined in Scheme S1, different BODIPYs (BODIPY 1, BODIPY 2 and BODIPY3) were prepared by the procedures reported by our group with some modifications.41,42 The structure of obtained products were validated via 1H NMR and MALDI-TOF mass spectrometry (Figure S1). Subsequently, the Suzuki coupling polymerization was carried out between DPP derivative M1 and BODIPY.43 The obtained polymers were repeatedly precipitated into methanol, and the CPs containing BODIPY 1, 2 and 3 was named as P0, P1 and P2, respectively. To calculate the Mw (molecular weight) and PDI (polydispersity index) of the polymers, gel permeation chromatogram in THF towards polystyrene standard was carried out. The Mw of P0-P2 were found to be 34.8, 46.9 and 20.1 kDa, respectively, and the PDI were around 2.16, 2.00 and 2.49, respectively (Figure S2a). In addition, the solubility of the final polymers was also enhanced in chloroform, THF and toluene by increasing the methyl groups on BODIPY (Figure S2b).

Figure 1. (a) Absorption spectra of P1 and P2 in CHCl3/THF/H2O mixed solution (39.5/39.5/1, v/v/v). (b) Absorption spectra of the CPs in toluene at different temperatures.


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To further verify the effect of BODIPY’s structure on the properties of CPs, photophysical characteristics were monitored by UV-vis-NIR absorption spectra. As revealed by Figure 1a and Figure S3a, P0 and P2 showed single peaks with maxima absorption (λmax) at 733 and 638 nm, respectively. By contrast, P1 displayed a wide absorption spectrum with λmax at 873 nm and strong shoulder at 800 nm. Moreover, we also measured UV-vis-NIR spectra at different temperature (Figure 1b and Figure S3b). The blue-shifted absorption and the decreased absorbance under continuous heating were mainly ascribed to the disaggregation of polymer chains at high temperature. In addition, the shoulder absorption still existed apparently even after increasing temperature, which might be attributed to the strong inter-chains aggregation effect within the polymers P1.44,45 In consideration of the UV-vis-NIR absorption spectra, P1 has the longest λmax, followed by P0 and finally P2, which was not consistent with our hypothesis. In theory, the fewer number of methyl groups at the β and β' position on BODIPY, the more planar structure and higher degree of conjugation is expected for the formed polymers, and the order of λmax should be P0, P1 and P2. In order to explain the phenomenon, density functional theory (DFT) calculations for the basic construction units were carried out using Gaussian 09 program reported by M. J. Frisch (Figure 2 and Table S1).30,46 For P0-P2, the theoretical LUMO (lowestunoccupied molecular orbital) energy were -3.15 eV, -2.98 eV and -2.77 eV, and the HOMO (highest-occupied molecular orbital) energy were -4.73 eV, -4.68 eV and -4.76 eV. The band gaps were calculated to be 1.58 eV, 1.7 eV and 1.99 eV for P0, P1 and P2, respectively (Figure 2a and Table S1). Interestingly, P0 had the lowest band gap value, which followed by P1 and P2. The calculated results of P1 and P2 were exactly in accordance with the actual results. Reducing of the number of the methyl substituents on BODIPY monomer resulted in enhanced planarization configuration of the final polymers, simultaneously with decreased energy band


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gaps and increased absorption peak. In Figure 2b and Table S1, the theoretical calculations of the repeating units of the CPs based on B3LYP/6-31G* level demonstrated P0 possessed the smallest inter-ring torsion angles and band gap, but P0 didn’t have the longest λmax. This anomalous deviation might be the absence of methyl substitution on BODIPY 1, which led to the difficulty in maintaining the planar and conjugation structure of P0 because of the free rotation in dissolved states.


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Figure 2. (a) LUMO and HOMO surface plots from DFT calculations for model compounds representing polymers (P0-P2). (b) Schematic illustration of construction units, and optimized spatial configurations of the representing units of P0-P2 from DFT calculations.


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Subsequently, in order to excavate the potential application of BODIPY-DPP CPs as PTAs and considering the solubility of the CPs, we selected P1 and P2 with absorption band at 873 and 638 nm as the examples, and made the nanoparticle formulation (P1 NPs and P2 NPs) by using amphiphilic polymer Pluronic F127 (Scheme 2). As showed by transmission electron microscopy (TEM), homogeneous spherical nanostructures could be observed in Figure S4a and c. The size, size distribution and surface potential of P1 NPs and P2 NPs were monitored via dynamic light scattering (Figure S4b, d and Figure S5). These results were in accordance with that obtained from TEM images. Because the fluorescence intensity of P1 and P2 were too low to be used for imaging (Table S2), we further encapsulated another high NIR-emissive BODIPY dye in the NPs (NIRP@F127 NPs) (Figure S4e and Figure S6). As exhibited in Figure S4f-g, the morphology and size don’t change after encapsulating cargo. The stability of NPs is of crucial importance for their practical bio-applications. Therefore, the size and size distribution of the NPs solution and PBS (pH 7.4) solution with 10% FBS were tested. In Figure S7, all the NPs could maintain favorable stability with negligible changes in simulated physiological environment.


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Scheme 2. Schematic presentation and applications of corresponding nanoparticles (P1 NPs and P2 NPs) as photothermal agents. The photophysical properties of P1, P2 and corresponding NPs were evaluated by UV-vis absorption spectroscopy. Figure S8 showed that P1 NPs and P2 NPs in water exhibited strong NIR absorption and the spectra were all red-shifted in comparison with their dissolved state in organic solution, which were caused by the J-aggregation in NPs.47 We studied the photothermal performance under laser irradiation (808 nm and 685 nm) with various concentrations and power density. As shown in images taken by infrared thermal camera (Figure 3a), the negative control groups demonstrated that the temperature of water increased by 5.8 oC and 1.8 oC from room temperature under 808 nm and 685 nm laser irradiation, respectively. However, temperature of the solutions significantly rose under the same condition in the presence of P1 NPs and P2 NPs


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with irradiation. After that, for the sake of calculating the photothermal conversion efficiencies (η) of P1 NPs and P2 NPs, we recorded temperature changes of the nanoparticles suspensions with irradiation and turn-off treatment (in Figure 3b-c(i)).48 The plot of cooling time versus -Lnθ of temperature changes without irradiation was displayed in Figure S9a-b. The calculated η of P1 NPs and P2 NPs were about 30.5% and 31.1%, respectively, which were high enough to induce fatal hyperthermia in malignant tumor cells. Moreover, the robust PTT stability of P1 NPs and P2 NPs were validated via cyclic heating-cooling experiments (Figure S9c-d). In Figure 3b-c(ii, iii), P1 NPs and P2 NPs also exhibited typical laser intensity and concentration-dependent behavior. In order to testify the photostability of P1 NPs and P2 NPs with continuous laser irradiation, ICG (indocyanine green) was performed as the contrast. From Figure S10, negligible UV-vis absorption reduction could be observed for P1 NPs and P2 NPs after warming-up experiment, while, the absorbance of ICG decayed dramatically. We could also obtain the identical conclusion intuitively by comparing the color changes of solutions of the three PTAs before and after laser irradiation (Figure S10).


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Figure 3. (a) Thermal images of P1 NPs and P2 NPs solutions with 808 nm laser irradiation at intensity of 0.57 W cm-2 and 685 nm laser irradiation at intensity of 0.55 W cm-2 at different times. Basic PTT performance of (b) P1 NPs and (c) P2 NPs. From left to right were: i: photothermal response of the nanoparticles under corresponding laser irradiation and 5 min later the laser were shut down; ii: PTT conversion behavior of the NPs by various laser power


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densities under fixed concentrations (25 µg mL-1); iii: NPs’ heating curves by deviating concentrations under irradiation along with time. The endocytosis behavior of NPs was investigated in human cervical carcinoma (HeLa) cells by CLSM (confocal laser scanning microscopy) and FCM (flow cytometry). NIRP@F127 NPs were utilized to monitor the cellular uptake process. As shown in Figure 4a and S12a, both CLSM and FCM depicted the similar results. Time-dependent cellular internalization of P1 NPs or P2 NPs NPs could be observed. In addition, compared with incubation at 37 oC, the uptake of P1 NPs or P2 NPs by HeLa cells decreased significantly at low temperature (4 oC), which is accorded with ATP-mediated endocytosis pathway. Furthermore, the detailed endocytosis way of the NPs was determined by co-localization of the NPs (NIRP@F127) and lysosomes marked with Lyso-Tracker Green. As shown in Figure S11, the yellow area was the overlay of green fluorescence from Lyso-Tracker Green and the red fluorescence from NIR-BODIPY, implying the accumulation of nanoparticles in lysosomes. The high η of P1 NPs or P2 NPs in vitro stimulated us to further explore their PTT therapeutic effect on tumor cells. MTT assays were implemented on HeLa cells to quantified the cytotoxicity of the NPs. In Figure 4b, P1 NPs and P2 NPs both displayed dose-dependent cellular toxicity against tumor cells with dramatic decline of cell viability after laser irradiation, while almost no tumor cells were killed without irradiation. The IC50 values were 6.65 and 11.63 μg mL-1 of P1 NPs and P2 NPs towards HeLa cells, respectively. Afterwards, calcein AM and propidium iodide (PI) co-staining experiment was carried out to obviously distinguish live (green fluorescence) and dead (red fluorescence) cells. In Figure S12b, both P1 NPs and P2 NPs could cause serious PTT injury and almost all tumor cells were killed under laser irradiation, which were also accompanied with both concentration and laser power intensity-dependence as evidenced via the strong red fluorescence. In contrary,


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cells from the control groups displayed green fluorescence, illustrating favorable cytocompatibility of the NPs without irradiation. Then, Annexin-V FITC/PI staining assays were applied for quantitatively determining the PTT effect of NPs. It could be seen in Figure 4c that no noticeable differences for cells treated with PBS with or without laser irradiation, suggesting ignorable effect on cell apoptosis of laser irradiation. However, significant apoptosis could be seen for the cells after incubation of P1 NPs and P2 NPs in the presence of laser irradiation and the apoptosis rates (Q2+Q3) of P1 NPs and P2 NPs were 75.98% and 53.81%, respectively.

Figure 4. (a) CLSM results of HeLa cells with incubation of NIRP@F127 NPs (NIR-BODIPY: 10 µg mL-1) at 37 oC for 0.5, 2 and 6 h and at 4 oC for 0.5 h and 2 h, respectively. Scale bars, 20 µm. (b) Cell viabilities of HeLa cells with treatment of P1 NPs and P2 NPs with and without


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irradiation. (c) FCM analysis of HeLa cells with treatments of P1 NPs (P1: 12.5 µg mL-1) and P2 NPs (P2: 25 µg mL-1), respectively. Photoacoustic imaging (PA) emerges as a novel non-invasive biomedical imaging strategy. As the results, the PA imaging capability of P1 NPs was used to investigate their biodistribution. Naked mice were intravenously (i.v.) injected with P1 NPs (1.5 mg kg-1) and the tumors’ photoacoustic signals were collected at different time points. From Figure 5a, we could observe the PA intensity at the tumor tissue appeared gradually, implying the effective accumulation of NPs at tumor, and the intensity reached the maximum value after 6 h. What’s more, we also used the NIRP@F127 NPs to describe the whole body biodistribution in vivo with the aid of nearinfrared fluorescence (NIRF) imaging systems. As demonstrating in Figure 5b-c, the amount of NPs in the tumor site increased gradually with maximal accumulation at 6 h post injection, which confirmed the most rational time for photothermal therapy. After that, we excised the main organs of mice at specified time points. As indicated in Figure 5c (above), majority of NPs were concentrated in liver and lung in the early stage of systematic injection. The fluorescence intensity of liver reached its maximum value at 0.5 h after administration and then slowly decreased to half of the original after 24 h. Meanwhile, the amount of NPs in lung increased gradually and the NIRF intensity reached the maximum 24 h post injection.


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Figure 5. (a) PA images of tumor obtained at specified time after being i.v. injected of P1 NPs at a dose of 1.5 mg kg-1. (b) NIRF images of mice with i.v. injection of NIRP@F127 NPs (NIRBODIPY: 1 mg kg-1, P1: 1.5 mg kg-1) at specified time periods. (c) NIRF pictures and fluorescence signals of main organs (From left to right were heart, liver, spleen, lung and kidney) and tumor extracted from mice intravenously injected with NIRP@F127 NPs at different time points. All the tumor is circled. In vivo photothermal tumor inhibition effect was estimated on tumor-containing female Kunming mice. The mice were randomly assigned to 11 groups with various treatments: 1: PBS; 2: 808 nm laser; 3: 685 nm laser; 4: intratumoral (i.t.) injection of P1 NPs; 5: i.t. injection pf P2 NPs; 6: intravenous injection of P1 NPs; 7: i.v. injection of P2 NPs; 8: i.t. injection of P1 NPs+808 nm laser irradiation; 9: i.v. injection of P1 NPs+808 nm laser irradiation; 10: i.t. injection of P2 NPs+685 nm laser irradiation; 11: i.v. injection of P2 NPs+685 nm laser irradiation. First of all, IR thermal camera was utilized to detect tumor temperature changes after the mice were administrated by P1 NPs and P2 NPs with irradiation. In Figure S13, the


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temperature of tumors manifested a rapid rise up to approximately 60 oC within the first 10 min irradiation when the mice were treated with i.t. injection. Under the same conditions, for the i.v. injected mice, temperature of tumor site increased slightly slowly because of limited accumulation of the NPs in the tumor. By contrary, negligible temperature increase was observed for the control group. During the PTT treatment process, tumor tissues were irradiated for one time. The body weight and tumor volumes were monitored every other day. In Figure 6a-b, mice with administration of P1 NPs and P2 NPs under laser irradiation, the size and weight of tumors were the smallest in comparison with other control ones, suggesting the prominent photothermal efficiency and cancer inhibition effect. According to Figure 6c, the body weight of mice increased steadily in 11 days, which suggested no obvious systemic toxicity of these two nanomaterials at current dose. Next, H&E and TUNEL staining images of excised tumor regions were collected to demonstrate the cellular damage and cellular apoptosis of tumors caused by photothermal therapy. From the images in Figure 6d, severe destruction of tumor cells could be detected in the groups with injection of P1 NPs and P2 NPs under laser irradiation while no distinct cell damage could be found for the control groups. Similar results could also be observed in the TUNEL staining images. As depicted in Figure S14, both P1 NPs and P2 NPs exhibited effective photothermal tumor killing ability regardless of the mode of injection administration, which were indicated by the strong green fluorescence from the CLSM images in comparision with the tumor slices from other groups. Furthermore, for the sake of assessing the biosafety of the nanoparticles, histological section of the main organs, whole blood panels and serum biochemistry were executed. As depicted in Figure S15, no significant differences were detected for the control groups and experimental groups with treatment of P1 NPs and P2 NPs, illustrating the great systemic biocompatibility of these nanoparticles.


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Figure 6. (a) Relative tumor sizes, (b) tumor photos, (c) relative body weight changes and (d) H&E staining of tumor sites of mice from different groups with treatment of 1: PBS, 2: 808 nm laser, 3: 685 nm laser, 4: i.t. injection of P1 NPs, 5: i.t. injection of P2 NPs, 6: i.v. injection of P1 NPs, 7: i.v. injection of P2 NPs, 8: i.t. injection of P1 NPs+808nm laser irradiation, 9: i.v. injection of P1 NPs+808nm laser irradiation, 10: i.t. injection of P2 NPs+685 nm laser irradiation, 11: i.v. injection of P2 NPs+685 nm laser irradiation. Scale bars: 200 µm. Statistical significance was assessed via one-way ANOVA test by SPSS, **P≤0.01, ***P≤0.001. CONCLUSION In conclusion, we have successfully demonstrated the fabrication of three CPs based on BODIPY and DPP, and the BODIPY monomers have different structures by varying the diverse amounts


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of methyl substitution. After experimental analysis and theoretical calculation, we generally concluded that less methyl substitution on BODIPY formed CPs with extended absorption wavelength because more planarization structure and higher degree of π-conjugation could lead to smaller band-gap energy of BODIPY-DPP CPs. Moreover, these CPs possess robust stability and strong absorbance at NIR region, which could be used as potential photosenitizers for PTT. The nanoparticle formulations exhibited good cytocompatibility, excellent photostability, high photothermal conversion efficacy, efficient photothermal activity toward cancer cells and effective photoacoustic imaging ability. Overall, this research provides a new method to develop versatile CPs through the molecular regulation, and demonstrates the great application prospects of BODIPY polymers as PTAs. EXPERIMENTAL SECTION Materials. The starting materials pyrrole was bought from Adamas Reagent Co., Ltd.. 2Methylpyrrolidine

and

2,4-dimethylpyrrole

were

purchased

from

Shanghai

Shuya

Pharmaceutical Technology Co., Ltd. and Suzhou Boke Chemistry Co., Ltd., respectively. 2,5Bis(2-octyldodecyl)-3,6-bis(5-(4,4,5,5-tetramethyl-1,3,2-diborate-2-yl)thiophene-2-yl)[3,4c]pyrrole-1,4(2H,5H)-dione (M1) was bought from Suzhou Nakai Technology Co., Ltd.. Calcein-AM/PI staining assay kit was purchased from Jiangsu KeyGEN Biotechnology Co., Ltd.. DAPI, Lyso Tracker Green and Annexin V-FITC apoptosis kit were bought from Shanghai Beyotime Biotechnology Co., Ltd.. DeadEndTM TUNEL System was obtained from Promega Corporation. The synthesis of monomers BODIPY 1, BODIPY 2 and BODIPY 3. Trifluoroacetic acid (25 μL) was added to 4-(hexadecyloxy)benzaldehyde (10.0 mmol, 3.47 g) and pyrrole (22.0 mmol,


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1.53 mL)/2-methylpyrrolidine (2.25 mL)/2,4-dimethylpyrrole (2.26 mL) in CH2Cl2. The mixture was stirred overnight, then DDQ (10.0 mmol, 2.27 g) was added. After 1 h, NEt3 (10 mL) and BF3•Et2O (12 mL) were successively added under ice bath cooling. Another 4 h of stirring later, the mixture was extracted with saturated Na2CO3 solution and then concentrated on a rotary evaporator.

Brown

oily

residue

was

depurated

by

silica

column

chromatography

(CH2Cl2:hexane=1:1). NIS (6.2 mmol, 1.4 g) was put into the solution of the product of the aforementioned step in chloroform and the mixture was stirred overnight. After the reaction, the purification process of the mixture was basically the same as the steps mentioned above. Deep red solids were obtained after purification by silica gel columns (CH2Cl2:hexane=1:1). General synthesis of polymers P0-P2: P0. BODIPY 1 (76 mg, 0.10mmol), M1 (111 mg, 0.10mmol), Pd(dppf)Cl2 (13 mg) and Na2CO3 (106 mg, 1.00 mmol) were put into a 250 mL flask with support. After that, the tube was extracted to vacuum and back-filled with argon about three times. Degassed solvent mixture of toluene/ethanol/water was transferred to the round-bottomed flask through a septum. Afterwards, the reaction was continued for 48 h under inert gas atmosphere at 85 oC. After completion, the organic solvent was eliminated. The mixture was dissolved in chloroform, and then slowly dropped into cold methanol with stirring. After 15 min of stirring, polymer was collected and the above processes were repeated several times. Synthesis of P1 and P2: Following the procedure for polymer P0, using BODIPY 2 and BODIPY 3.


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Preparation of NPs. Preparation of the nanoparticles were based on the nanoprecipitation method.38 For the NIRP@F127 NPs, the difference was dissolving NIR-BODIPY and P1 in THF at the same time and the subsequent process was the same as the preparation method of P1 NPs. In vitro PTT effects of NPs. P1 NPs and P2 NPs dispersions with varying concentrations were treated with an 808 nm laser at intensity of 0.57 W cm-2 and 685 nm laser at intensity of 0.55 W cm-2, respectively. Temperatures changes were recorded every 10 s. The photothermal response of P1 NPs and P2 NPs solutions (25.0 µg mL-1, 200 μL) was characterized with and without irradiation, and temperatures were also recorded every 10 s. Photothermal conversion efficiency (η) was counted according to previous strategy.40 Cycling heating-cooling curves were investigated under successive 808 and 685 nm laser illumination. To detect the photostability of nanoparticles under irradiation, ICG with the same concentration was used as the contrast. Briefly, 3 mL of NPs solutions and ICG (50.0 µg mL-1) were illuminated continuously for 10 mins with 800 and 685 nm laser, then the absorption spectra of corresponding substances before and after irradiation were detected. Cell experiments. All the basic cellular experiments were provided by our previous works.38,49 The difference was that the materials were replaced by corresponding P1 NPs, P2 NPs or NIRP@F127 NPs. Animals and tumor model. All the animals were treated based on the regulation of Research Animals proposed by Jilin University Studies Committee. All the Kunming mice were subcutaneously injected with U14 cells into the armpits. NIRF imaging and bio-distribution of CPs NPs. NIRP@F127 NPs (NIR-BODIPY: 1 mg kg-1, P1 NPs: 1.5 mg kg-1) was i.v. injected into the mice and then the whole body NIRF images were


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achieved using intravital NIR imaging system (Meatro 500FL) with deep red (671-705 nm) light excitation. Under anesthesia, the mice were at different time period post injection, respectively. Tumor-containing Kunming mice were i.v. treated with NIRP@F127 NPs and the concentration of NIR-BODIPY and P1 were 1 mg kg-1 and 1.5 mg kg-1, respectively. The main organs and tumors were collected and imaged at 0.5, 2, 6, 12, 24 and 48 h to determine the bio-distribution of CPs NPs. PA imaging. PA imaging experiments were performed as reported article.38 Tumor-bearing nude mouse was treated by i.v. injection of P1 NPs at a dose of 1.5 mg kg-1. Photoacoustic images were recorded at 2, 6, 18, 24, 30 and 48 h, and then analyzed by ViewMOST software. Evaluation of therapeutic efficacy in vivo. Tumors were allowed to grow to approximately 100 mm3. Afterwards, these mice were randomly allocated to 11 groups for different formulations (n=3): (1) injection of PBS or only laser irradiation (808 and 685 nm); (2) i.v. and i.t.injection of P1 NPs+808 nm laser irradiation (1.5 mg kg-1); (3) i.v. and i.t.injection of P2 NPs+685 nm laser irradiation (1.5 mg kg-1); (4) i.v. and i.t. injection of P1 NPs and P2 NPs without laser irradiation. The whole illumination process was executed only once and the time was lasted for 20 min. After that, tumor lengths and widths were measured every day after treatment. Tumor sizes were determined by the formulation: length×width2/2 (mm3), and the body weight was also recorded. Systemic toxicity study of the NPs. Three Kunming mice were used with intravenous injection with the nanoparticles at a dose of 1.5 mg kg-1 for each group. 11 days later, liver/kidney function markers and hematology analysis were obtained. TUNEL immunofluorescence staining. The previous sample preparation process was similar to H&E staining, which was followed by staining with TUNEL staining kit.


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ASSOCIATED CONTENT Supporting Information. Synthesis routes of P0-P2; 1H NMR and MALDI-TOF spectra of BODIPY monomers; GPC, Mw, Mn and polydispersity of polymer P0-P2; Absorption spectra of P0 and P0 in toluene at varying temperatures; Solubility of CPs in different solvents; Illustration of fabrication NIRP@F127 NPs; DLS result and TEM image of NIRP@F127 NPs; Zeta potential of different NPs; Fluorescence quantum yields of different substances; Photophysical of NIR-BODIPY solution; Size and PDI changes of NPs and in PBS with 10% FBS; Absorption spectra of P1-P2 solutions and P1 NPs and P2 NPs solutions; Cooling time versus -Lnθ of NPs; Heating reproducibility of NPs solutions; FCM data of NIRP@F127 NPs; Fluorescent images of PI and calcein AM costained HeLa cells with incubation with NPs; Temperature increase of tumors in different groups; TUNEL staining of tumor slices; H&E staining of main organs, hematology data and serum biochemical analysis. AUTHOR INFORMATION Corresponding Author Email:

[email protected]

Acknowledgement We are very thankful for Dr. R. Zhao assisted in the theoretical calculation of CPs and Prof. C. Dou supervised the research project. Funding Sources We are very grateful for the financial support from the National Natural Science Foundation of China (Project No. 51773197).


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Rational Design of BODIPY-Diketopyrrolopyrrole Conjugated

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