Zinc(II) Metalated Porphyrins as Photothermogenic Photosensitizers

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Zinc (II) Metalated Porphyrins as Photothermogenic Photosensitizers for Cancer Photodynamic/Photothermal Synergistic Therapy Kaikai Ding, Ye-Wei Zhang, Weili Si, Xiangmin Zhong, Yu Cai, Jianhua Zou, Jinjun Shao, Zhou Yang, and Xiaochen Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15583 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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ACS Applied Materials & Interfaces

Zinc (II) Metalated Porphyrins as Photothermogenic Photosensitizers for Cancer Photodynamic/Photothermal Synergistic Therapy Kaikai Ding,a‡ Yewei Zhang,c‡ Weili Si,a Xiangmin Zhong,b Yu Cai,a Jianhua Zou,a Jinjun Shao,a* Zhou Yang,b* Xiaochen Donga* a

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

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing (China) E-mail: [email protected]; [email protected] b

Beijing Key Laboratory of Function Materials for Molecule & Structure Construction,

School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing (China) E-mail: [email protected] c

Department of Hepatobiliary and Pancreatic Surgery, Zhongda Hospital, Medical School,

Southeast University, Nanjing (China) ‡

These authors contributed equally

Abstract Porphyrin derivatives are the first-generation photosensitizers, and to design a strong NIR-absorbing porphyrin with good water solubility is highly desired for better therapeutic effect to treat tumors. Herein, three new porphyrin derivatives, 5,10,15,20-tetrakis(3,4dimethoxyphenyl) porphyrin (P1), 5,10,15,20-tetrakis(3,4-dimethoxyphenyl) Zinc porphyrin (ZnP1), and 5,15-bis (3,4-dimethoxyphenyl)-10,20-bis ((4-methoxyphenyl)ethynyl) Zinc porphyrin (ZnP2) have been synthesized. Among them, ZnP2 shows the longest and most 1

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intensive Q-bands in the near-infrared (NIR) region, as endows it the strongest light-harvesting capability and deepest tumor tissue penetration. The three porphyrin derivatives were prepared into nanoparticles (NPs) via nanoprecipitation method, and the NPs exhibit good water dispersibility and passive tumor-targeting property through enhanced permeability and retention (EPR) effect. Furthermore, these NPs demonstrate both photodynamic and photothermal effects. Through systematical study of the singlet oxygen quantum yield and cytotoxicity of P1, ZnP1 and ZnP2 NPs in vitro on Hela cells, it is found that ZnP2 shows the highest singlet oxygen quantum yield (79%) and its NPs show the best therapeutic efficacy in vitro. In vivo experiments disclosed that ZnP2 NPs present high phototoxicity, low dark toxicity and excellent bio-compatibility, and could be used as a promising photothermogenic photosensitizer for cancer treatment.

KEYWORDS: porphyrin, photosensitizer, NIR absorption, photodynamic therapy, photothermal therapy

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INTRODUCTION Over the past two decades, cancer has become the major threat to human beings’ wellness around the world, and considerable efforts have been devoted to the cancer treatment.1-2 Phototherapy, an emerging light induced therapeutic modality, usually contains photodynamic therapy (PDT) and photothermal therapy (PTT).3-4 Photodynamic therapy (PDT) is a promising cancer treatment approach with the advantages of low toxicity and non-invasive characteristics.5-8 During the PDT process, light energy is absorbed by the photosensitizer,9-11 in which an electron will be promoted from ground state (S0) to singlet state (S1), and then converted to triplet state (T1) through intersystem crossing (ISC) to react with surrounding oxygen,12-13 and producing reactive oxygen species (ROS, mainly singlet oxygen 1O2), to destroy tumor cells.14 Photosensitizer is the key factor for PDT, which usually requires high ROS generation yield, good water solubility, and good biocompatibility.15-16 However, current photosensitizers used in clinic are mostly free molecules with the disadvantages of low bioavailability and bad water solubility, which only offer poor therapeutic effects, even can bring side effects in the body system.17 Therefore, to design photosensitizers with high ROS generation and specific tumor-targeting property is strongly imperative for PDT.18-19 Photothermal therapy (PTT) is another widely attractive phototherapy for cancer treatment, which can kill tumor cells with high effectiveness, high selectivity, and low side-effects by laser-induced hyperthermia.20-23 Currently, most of the photothermal agents are inorganic nanomaterials (e.g. Noble metal nanoparticles, transition metal sulfide nanosheets, graphene sheets, and carbon nanotube derivatives) because of their high photothermal conversion efficiency (PCE) and excellent absorbance in the near-infrared (NIR) region,24-25 but the clinical use of these materials are limited by the long-term cytotoxicity, and poor body 3



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clearance.26 As a result, to develop the NIR dye based organic nanoparticles (NPs) with intense NIR absorption and high PCE is promising to substitute the inorganic nanomaterials, due to the great biocompatibility, easy to be metabolized in body, favorable photostability and thermal-stability.27-28 Porphyrin derivatives as the first kind of typical photosensitizers have been approved by the multinational governments drug regulatory authorities and have been widely applied in clinic due to their unique photodynamic and photothermal conversion capability.57-58 In addition, they have the advantages of low toxicity,29 good biocompatibility and low side effects to the human body.30 Nevertheless, there are still some problems of porphyrin derivatives, which limits their further use, such as low absorption in NIR region (e.g. protoporphyrin IX and hematoporphyrin derivatives), deficient photostability (e.g. talaporfin and chlorin e6), poor water solubility, and no specific targeting to tumors.31-32 Therefore, rational design of a porphyrin based photothermogenic photosensitizer with good NIR absorption and high singlet oxygen quantum yield (QY) is very desirable for cancer photodynamic/photothermal synergistic therapy.33-37 Herein, three porphyrin derivatives, 5,10,15,20-tetrakis (3,4-methoxyphenyl) porphyrin (P1), 5,10,15,20-tetrakis (3,4-methoxy phenyl) zinc porphyrin (ZnP1), and 5,15-bis (3,4methoxyphenyl)-10, 20-bis(4-methoxyphenylethynyl) zinc porphyrin (ZnP2) have been designed and synthesized (Scheme 1). ZnP2 show the most intensive Q-bands absorption in the NIR region, and the highest singlet oxygen QY of 79% (43% for P1, 54% for ZnP1). Compared to P1, ZnP1 and ZnP2 introduced metal zinc can greatly enhance its photodynamic effect. The three porphyrin derivatives were prepared into nanoparticles (NPs) via nanoprecipitation method.38 And ZnP2 NPs also show a good photothermal effect with 4


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the efficiency of ~33.4%, which is higher than many photothermal agents reported.39 Both in vitro and in vivo results indicate that ZnP2 NPs have not only high phototoxicity, but also good biocompatibility, indicating ZnP2 is a promising photothermogenic photosensitizer for cancer photodynamic/photothermal synergistic therapy in clinic. RESULTS AND DISCUSSION The synthetic route for porphyrins P1, ZnP1 and ZnP2 is shown in Scheme 1. Porphyrin P1 was synthesized by reacting the aldehyde 1 with pyrrole 2 in DCM with subsequent addition of BF3•Et2O, and followed by oxidizing with DDQ. Later Zinc acetate was used to achieve the Zinc metalated porphyrin ZnP1 in an excellent yield of 98%.40 Porphyrin 4 was prepared between aldehyde 1 and dipyrrole 3, with following addition of BF3•Et2O and DDQ, then 4 was brominated with N-bromosuccinimide (NBS) in chloroform to produce 5. Metalloporphyrin 6 was obtained in a quantitative yield from 5 via using Zn(OAc)2 as metalation reagent. Palladium catalyzed Sonogashira-Hagihara coupling between 6 and the alkyne 7 to give ZnP2 with a good yield of 73%.41 1H NMR spectroscopy and MALDI-TOF mass spectrometry were used to identify the chemical structures and purity of all the compounds (Experimental Section and Supporting Information). The UV-vis absorption and fluorescence emission spectra of P1, ZnP1 and ZnP2 were recorded in DCM (Figure 1a, Figure 1b and Table 1). All porphyrins P1, ZnP1 and ZnP2 show intense Soret bands with respective peak maximum at 426, 432 and 459 nm, which corresponding to S0→S2 transition. P1 shows four weak Q bands with the absorption maximum of 521, 554, 598, and 653 nm. With Zinc metalation in the porphyrin center, the increased molecule symmetry reduces the number of Q-bands from four to two, and the Q-bands are resulting from S0→S1 transition. As a result, Zinc-metalated porphyrin ZnP1 5



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shows two weak Q bands at 563 and 605 nm; while Zinc-metalated porphyrin ZnP2 shows an intense peak with a maximum at 668 nm and a shoulder peak with a maximum at 610 nm in the Q region. Figure 1c presents the fluorescence emission spectra of P1, ZnP1 and ZnP2 in DCM. Two intense emission peaks are observed for both P1 and ZnP1; however, an intense peak at 709 nm, a shoulder peak at 760 nm and many fine peaks are observed for ZnP2 upon excitation at 488 nm. The P1, ZnP1 and ZnP2 NPs were prepared through nanoprecipitation method. THF solution of porphyrin (P1, ZnP1 or ZnP2) was quickly injected into water under sonication, after bubbling for 5 minutes to remove THF, a transparent solution was obtained with hydrophilic NPs inside.42 When the porphyrins were prepared into water-soluble NPs, their absorbance profiles become a little broader (Figure S1). In addition, a bathochromic-shift of around 10 nm could be observed, as indicating the intermolecular π-π stacking interactions. In comparison with P1 and ZnP1 NPs, the Soret bands of ZnP2 NPs show the lowest molar extinction efficient, nevertheless, the Q-bands of ZnP2 NPs at 668 nm present the largest molar extinction efficient (ε) of 0.23 × 105 M-1 cm-1, as indicates the potential of ZnP2 to be used as a good photothermal agent. The morphological dimensions and ultrastructure of P1, ZnP1 and ZnP2 NPs were studied by transmission electron microscope (TEM) and dynamic light scattering (DLS) (Figure S3). As shown in Figure 1e, TEM image shows that ZnP2 NPs exhibit good spherical morphology with a diameter approximately of 50-200 nm, as is satisfactory for passive tumor targeting since EPR effect.43-44 And the DLS measurement further indicates that ZnP2 NPs present relatively uniform size dispersion in deionized water with an average size of 158 nm (Figure 1f). The TEM and DLS results of P1 and ZnP1 NPs are shown in Fig. S3, both of 6


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them show the diameters ranging from 50 to 150 nm, which is similar to ZnP2 NPs. Besides, after ZnP2 NPs standing in aqueous solution for two days, there is no precipitation (Figure S16), confirming the good dispersion stability of ZnP2 NPs. Reactive oxygen species (ROS) are the key cytotoxic substances during PDT, and singlet oxygen (1O2) is the most prominent one of ROS.45-46 To confirm the photodynamic effect of P1, ZnP1 and ZnP2, the singlet oxygen quantum yields (Φ∆) were quantitatively measured by taking 9,10-anthracenediyl bis(methylene) dimalonic acid (ABDA) as the trapping reagent,47 and methylene blue (Φ∆ = 0.52 in CH2Cl2) as the reference.48 As shown in Figure 2a and Figure S4, it can be seen clearly that under the irradiation of Xe lamp, the absorbance of ABDA probe degraded much faster in ZnP2 solution than that in P1 and ZnP1 solution. As implies that ZnP2 present better ROS generation capability than P1 and ZnP1. Figure S5 shows the degradation slopes of ABDA probe within the absorbance range of 0.8-1.1 in CH2Cl2 and R2 is above 0.998, which suggests a linear degradation. According to singlet oxygen quantum yield calculation formula, the singlet oxygen quantum yields of P1, ZnP1 and ZnP2 are determined to be 43%, 54% and 79%, respectively (Table 1, Figure 2b, Figure S5).48 ZnP2 shows the highest singlet oxygen quantum yield, this is probably attributed to the prolonged conjugation length of the alkyne units. Furthermore, singlet oxygen sensor green (SOSG) was used as ROS probe to detect the photodynamic effect of ZnP2 NPs.59 As shown in Figure S12, ZnP2 NPs have a better photodynamic effect than that of Methylene Blue. Afterwards, the photothermal effect of P1, ZnP1 and ZnP2 NPs were investigated. As shown in Figure 3a, ZnP2 NPs exhibit the best photothermal effect at the concentration of 80 µg/mL under 660 nm laser irradiation, while P1 and ZnP1 NPs show only weak photothermal effect at the same concentration as ZnP2 NPs does; this is in good agreement with the molar 7



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extinction efficient of the Q bands at 660 nm. The photothermal effects of ZnP2 NPs at various concentrations from 20 µg/mL to 100 µg/mL were examined under 660-nm laser irradiation at a power density of 0.75 W/cm2 (Figure 3b), as indicating a good concentration dependence. As shown in Figure 3b, the temperature of 20 µg/mL ZnP2 NPs increased from 18 to 44 oC upon laser irradiation for 15 minutes. While the temperature of pure deionized water only increased from 18 to 22 oC under the same circumstances. The photothermal conversion efficiency (PCE) of P1, ZnP1 and ZnP2 NPs was also measured.49-50 PCE of P1 NPs is calculated to be 16.8%, which is relatively low (Figure S6). For ZnP1 NPs, which show almost no absorbance at 660 nm, as a result, its PCE could not be calculated quantitatively. PCE of ZnP2 NPs was calculated to be ~33.4%, which is slightly better than the traditional inorganic nanomaterials such as Au nanorods (21%), Cu9S5 (25.7%) (Figure 3c, Figure S6).51-53 The photothermal stability of ZnP2 NPs was tested as well, with the concentration of 150 µg/mL for five alternate on/off laser irradiation cycles. As shown in Figure 3d, ZnP2 NPs exhibits excellent photothermal stability even in the case of continuous laser irradiation for 50 minutes. Therefore, ZnP2 NPs could serve an efficient therapeutic agent for photodynamic/photothermal synergistic therapy. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to evaluate the biocompatibility and phototoxicity of P1, ZnP1 and ZnP2 NPs.54 As shown in Figure 4, all the NPs show excellent biocompatibility in the dark, and great phototoxicity under illumination. All P1, ZnP1 and ZnP2 NPs present a low cytotoxic effect on Hela cells in the darkness even at a relatively high concentration of 15 µg/mL (Figure S7). At the meantime, it can be found that the cell viability obviously decreases under the irradiation of Xe lamp for 8 minutes, and the half-maximal inhibitory concentration (IC50) for P1, ZnP1 8


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and ZnP2 NPs is determined as 7.8, 5.7 and 3.6 µg/mL, respectively, which is consistent with singlet oxygen quantum yield and photothermal conversion efficiency measured above. All the results demonstrate the efficient synergistic photodynamic/photothermal performance of ZnP2 NPs in tumor cells. The upper half of Figure 5 shows cellular uptake of ZnP2 NPs (3.6 µg/mL, 200 µL). Obviously, the red fluorescence demonstrates ZnP2 NPs are bounteously dispersed in the cytoplasm of Hela cells, meaning good uptake of ZnP2 NPs in tumor cells. In order to detect the ROS production in vitro, 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used as an ROS probe, while reacting with ROS, non-fluorescent DCFH-DA can be converted to fluorescent 2',7'-dichlorofluorescein (DCF).55 As shown in the lower half row of Figure 5, bright green fluorescence was observed due to the ROS generation upon excitation at 488 nm, which is ascribed to the highest singlet oxygen quantum yield of ZnP2. The cellular uptake and cellular ROS of P1 and ZnP1 are displayed in Figure S8 and Figure S9, respectively, which can also well enter the Hela cells and produce high ROS. From the in vitro results above, ZnP2 NPs were employed to investigate the photodynamic/photothermal therapeutic efficacy in vivo. ZnP2 NPs (50 µg/mL, 100 µL) were injected into the Hela tumor-bearing mice through tail vein (n = 5). Figure 6a shows the photothermal images of tumor site after intravenous injection of ZnP2 NPs to mice at different time. After 4 h, photothermal effect reaches the maximum at the tumor sites (laser irradiation for 3 minutes), indicating the excellent tumor targeting of ZnP2 NPs by EPR effect in vivo. Figure 6b shows the fluorescence imaging and tissue distribution of ZnP2 NPs in vivo. We can observe that the fluorescent drug signals at the tumor site reach the strongest at 4-6 hours. And after 24 h, ZnP2 NPs still mainly remained at the tumor tissue sites, which 9



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further indicates the excellent EPR effect in vivo. As shown in Figure 6b, after intravenous injection of ZnP2 NPs (50 µg/mL, 100 µL) for 48 h, almost no fluorescence signal can be detected in tumor-bearing mice, which means the body clearance time is about 48 h after treatment. The tumor volume and body weight change were recorded every two days. As shown in Figure 6c, the tumor volume of the control group and the no illumination group increased sharply as time went on, while the tumor volume of the illumination group (660 nm laser, 0.75 W/cm2) decreased for the first 6 days and left only one black scab for the next 10 days. After treatment for 8 times (16 days), the tumors of the mice of the illumination group disappeared. We still keep these mice for another 7 days, no obvious tumors were observed at all, indicating the tumors were completely cured and no tumor recurrence. Figure 6d shows the body weight change of these three groups, the mice in control group became thinner and the other two groups became fatter, implying the low dark toxicity and good bio-compatibility of ZnP2 NPs. After the treatments, these mice were sacrificed for the possible pathomorphology analysis of the major organs (heart, liver, spleen, lung and kidney).56 As shown in Figure 7, the hematoxylin and eosin (H&E) stained slices indicate that the treatment causes no obvious tissue damage or adverse effect in major organs. These results further demonstrate that ZnP2 NPs are non-toxic to normal organs and can efficiently destroy tumor cells. Therefore, these in vivo experiments prove that ZnP2 NPs can serve an efficient photothermogenic photosensitizer for cancer therapy. CONCLUSION In summary, three porphyrin P1, ZnP1 and ZnP2 have been successfully synthesized, which 10


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are made to NPs via nanoprecipitation to improve their water solubility and passive tumor-targeting property. All of P1, ZnP1 and ZnP2 have excellent photodynamic effect with high singlet oxygen quantum yield of 43%, 54% and 79%, respectively. More importantly, ZnP2 NPs show a good photothermal conversion efficiency (~33.4%). MTT assay and cell imaging results based on Hela cells indicate that ZnP2 NPs have good biocompatibility and phototoxicity. In vivo study further demonstrates that ZnP2 NPs has excellent PDT/PTT efficacy, and as can be used as a promising photothermogenic photosensitizer for cancer treatment.

EXPERIMENTAL SECTION Chemicals and Characterization 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (MO, USA). All the raw materials for synthesis were purchased from Adamas (China) and used without further purification. The 1H-NMR spectra were recorded on Bruker DRX NMR spectrometer (500 MHz) in CDCl3 or DMSO with tetramethylsilane (TMS) as the internal standard. Absorption spectra were recorded on an UV-3600 UV-Vis-NIR spectrophotometer (Shimadzu, Japan). Fluorescence spectra were measured on an F-7000 spectrometer (HITACHI, Japan). All experiments were performed in compliance with the relevant laws and institutional guidelines, and the institutional committee(s) has approved the experiments. Synthesis of 5,10,15,20-tetrakis (3,4-methoxy phenyl) porphyrin (P1) 3,4-dimethoxy benzaldehyde (1.660 g, 10 mmol) and fresh pyrrole (0.670 g, 10 mmol) were put into a 500-mL round bottom flask, then dry dichloromethane (250 mL) was added, 11



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ventilation with argon for 10 min to remove oxygen. After that, boron trifluoride diethyl ether (2.0 mL) was added and stirred for 3 h at room temperature. Later, DDQ (2.500 g) was added, and the reaction was stirred for another 30 min. After the completion of the reaction, the solvent was removed under reduced pressure. The crude product was purified by column chromatography (DCM: methanol = 100: 1) to give compound P1 as a purple-red solid (257 mg, yield 12%). 1H NMR (DMSO, 500 MHz): δ ppm 8.93 (s, 8 H); 7.78 (d, J = 7.5 Hz, 4 H); 7.28 (s, 4 H); 4.20 (s, 12 H); 3.98 (s, 12 H); -2.71 (s, 2 H, N-H). MALDI-TOF MS: m/z calcd for C52H46N4O8, 855.7, found 855.9.

Synthesis of 5,10,15,20-tetrakis (3,4-methoxy phenyl) zinc porphyrin (ZnP1) Compound P1 (257 mg, 0.3 mmol) and zinc acetate (146.8 mg, 0.8 mmol) were dissolved in a solvent mixture of dichloromethane and methanol (v/v = 9:1, 50 mL), the reaction was carried out in a single-necked round bottom flask at room temperature for 2 hours. Then the solvent was removed under reduced pressure, and the residue was purified with column chromatography (pure DCM as eluent) to obtain ZnP1 as a pure purple-red solid (270 mg, 98%). 1H NMR (DMSO, 500 MHz): δ ppm 9.01 (s, 8 H); 7.78 (s, 4 H); 7.75 (d, J = 8.9 Hz, 4 H); 7.25 (d, J = 4.8 Hz, 4 H); 4.10 (d, J = 5.0 Hz, 24 H). MALDI-TOF MS: m/z calcd for C52H44N4O8Zn, 918.4, found 919.7. Synthesis of 5,15-bis (3,4-methoxyphenyl) porphyrin (4) Dipyrrole (1.460 g, 10 mmol) and 3,4-dimethoxybenzaldehyde (1.660 g, 10 mmol) were dissolved in dichloromethane (150 mL) and placed in a flame-dried 500-mL round bottom flask, then wrapped with aluminum foil, ventilation with nitrogen to remove oxygen for 10 min at room temperature, the reaction was carried out in dark for 15 min. the 5 drops of boron 12


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trifluoride ether was added, after stirring for another 17 h, DDQ (3 g, 13.4 mmol) was added and stirring for another 30 min. After completion of the reaction, the solvent was removed under reduced pressure to give a dark solid which was rinsed with methylene chloride to give the crude product. The crude product was purified by column chromatography (DCM: methanol = 100: 1) to give 4 as a pure purple red solid (356 mg, 11.4%). 1H NMR (CDCl3, 500 MHz): δ ppm 10.34 (s, 2 H); 9.42 (d, J = 5.0 Hz, 4 H); 9.16 (d, J = 5.0 Hz, 4 H); 7.85 (d, J = 10.0 Hz, 4 H); 7.34 (s, 2 H); 4.24 (s, 6 H); 4.07 (s, 6 H); -3.02 (s, 2 H, N-H). MALDI-TOF MS: m/z calcd for C36H30N4O4 582.2, found 582.4.

Synthesis of 5,15-bis (3,4-methoxyphenyl) -10,20-dibromo porphyrin (5) Compound 4 (233 mg，0.4 mmol) and NBS (178 mg, 1.0 mmol) were dissolved in 40 mL of chloroform and placed in a 100 mL round bottom flask, then 0.16 mL of pyridine was added dropwise and reacted at 0 ℃ for 3 h. The solvent was removed by distillation under reduced pressure and purified by column chromatography using dichloromethane as eluent to obtain a purple solid (255 mg, 86%). 1H NMR (CDCl3, 500 MHz): δ ppm 9.64 (d, J = 5.0 Hz, 4 H); 8.96 (d, J = 5.0 Hz, 4 H); 7.72 (d, J = 10.0 Hz, 4 H); 7.44 (s, 2 H); 4.22 (s, 6 H); 4.05 (s, 6 H); -2.71 (s, 2 H, N-H). MALDI-TOF MS: m/z calcd for C36H28Br2N4O4 740.0, found 740.2. Synthesis of 5,15-bis (3,4-methoxyphenyl)-10,20-dibromo zincporphyrin (6) Compound 5 (222 mg, 0.3 mmol) and zinc acetate (147 mg, 0.8 mmol) were dissolved them in 50 mL of dichloromethane and methanol mixed solvent (v/v = 9:1), the reaction was carried out in a single-necked round bottom flask at room temperature for 2 hours. After the reaction was finished, the solvent was removed under reduced pressure, and purified with a column chromatography (pure DCM as an eluent) to obtain a pure purple-red solid (236 mg, 13



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98%). 1H NMR (CDCl3, 500 MHz): δ ppm 9.64 (d, J = 5.0 Hz, 4 H); 8.96 (d, J = 5.0 Hz, 4 H); 7.72 (d, J = 10.0 Hz, 4 H); 7.44 (s, 2 H); 4.22 (s, 6 H); 4.05 (s, 6 H). MALDI-TOF MS: m/z calcd for C36H22Br2N4O4Zn 740.0, found 802.2. Synthesis

of

5,15-bis(3,4-methoxyphenyl)-10,20-bis(4-methoxyphenylethynyl)

zinc

porphyrin (ZnP2) Compound 6 (80.2 mg, 0.1 mmol) and 4-methoxyphenyl acetylene (52.8 mg, 0.4 mmol) were dissolved in a mixture of THF and Et3N (v/v = 3:1, 20 mL) in a 100 mL round bottom flask, bubbling nitrogen for 30 min. After that, PdCl2(PPh3)2 (10.5 mg, 0.015 mmol) and cuprous iodide (7.6 mg, 0.04 mmol) were added to the reaction system, then stirring in dark for 4 h under reflux. After the reaction was cooled, the solvent was removed under reduced pressure. The product was purified by column chromatography using dichloromethane: methanol = 100: 1 (v/v) as an eluent to give an ink green solid (66.0 mg, 73%). 1H NMR (CDCl3, 500 MHz): δ ppm 9.71 (d, J = 5.0 Hz, 4 H); 8.97 (d, J = 5.0 Hz, 4 H); 7.92 (s, 2 H); 7.75 (d, J = 10.0 Hz, 8 H); 7.09 (d, J = 5.0 Hz, 4 H); 4.23 (s, 6 H); 4.03 (s, 6 H); 3.91 (s, 6 H). MALDI-TOF MS: m/z calcd for C54H40N4O6Zn 904.2, found 904.3. ASSOCIATED CONTENT Supporting Information Supporting Information available: Experimental details, normalized UV-vis absorbance of P1, ZnP1 and ZnP2 in DCM and their corresponding NPs in water (Figure S1), photostability measurements of P1, ZnP1 and ZnP2 in DCM (Figure S2), DLS size distribution and TEM images of P1 NPs and ZnP1 NPs (Figure S3), the degradation of ABDA under Xe lamp irradiation of Methylene Blue, P1, ZnP1, and ZnP2 (Figure S4), linear fitting of the degradation of ABDA (Figure S5), alternate temperature rising and dropping curve and linear 14


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fitting of P1 NPs and ZnP2 NPs (Figure S6), biocompatibility of P1, ZnP1 and ZnP2 NPs at various concentrations in dark (Figure S7), cellular uptake and ROS of P1 NPs and ZnP1 NPs in Hela cells (Figure S8 and Figure S9), different groups of nude mice, tumors and tumor slice (Figure S10), the detect of singlet oxygen with singlet oxygen sensor green (Figure S12), MTT assay with 660 nm laser (Figure S13), the stability of ZnP2 NPs (Figure S13-15). ACKNOWLEDGMENTS This work was supported by NNSFC (61525402, 61775095, 61604071, 61371066), Jiangsu Provincial key research and development plan (BE2017741, 2015720), the Key University Science Research Project of Jiangsu Province (15KJA430006), Natural Science Foundation of Jiangsu Province (BK20161012).

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Scheme 1. Synthetic routes for compounds P1, ZnP1 and ZnP2.

22


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Figure 1. (a) Normalized UV-vis absorbance of P1, ZnP1 and ZnP2 in DCM; (b) UV-vis absorbance of P1, ZnP1 and ZnP2 NPs in deionized water; (c) Normalized fluorescence spectra of P1, ZnP1 and ZnP2 in DCM; (d) The photographs of P1, ZnP1 and ZnP2 NPs in deionized water (c = 80 µg/mL); (e) The TEM image of ZnP2 NPs; (f) The DLS size distribution of ZnP2 NPs.

23



1.2

0.8

1.2 0 min 1 min 2 min 3 min 4 min 5 min 6 min 7 min

Absorbance

1.0

Absorbance

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

Page 24 of 31

y = -0.09x + 1.145 R2 = 0.998

1.1

1.0

0.9

0.6 0.8

0.4 320

340

360

380

400

420

0

Wavelength (nm)

1

2

3

4

Time (min)

Figure 2. (a) The degradation of ABDA under Xe lamp irradiation of ZnP2; (b) Linear fitting of the degradation of ABDA in ZnP2 solution.

24


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Figure 3. (a) The photothermal of P1, ZnP1 and ZnP2 NPs under 660 nm laser (c = 80 µg/mL, P=0.75 W/cm2 ); (b) The photothermal effect of ZnP2 NPs under the laser in 660 nm with different concentrations (P=0.75 W/cm2); (c) The graph is the temperature rising and dropping curve of ZnP2 NPs (c = 80 µg/mL, P=0.75 W/cm2); (d) The photothermal stability test of ZnP2 NPs for five alternate on/off laser irradiation cycles (c = 150 µg/mL, P=0.75 W/cm2).

25



1.0

0.8

0.6

Darkness Illumination

0.4

0

2

4

1.0

0.8 0.6


0.4

6

8

Concentration (µg/mL)

10

Cell Viability

Cell Viability

1.0

Cell Viability

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

Page 26 of 31

0.8 0.6


0.4

0.2

0.2

-1

0

1

2

3

4

5

6


7

8

0

1

2

3

4


5

Figure 4. Cell viability of Hela cells treated with different concentrations of P1 NPs (a), ZnP1 NPs (b), and ZnP2 NPs (c) with and without light illumination.

26


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Figure 5. Cellular uptake of ZnP2 NPs in Hela cells at the concentration of 3.6 µg/mL, and the ROS generation in Hela cells with DCFH-DA as a probe. (left panel, DAPI; middle panel, fluorescence image; right panel, merged image, and the scale bar is 20 µm).

27



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Figure 6. (a) Photothermal imaging in tumor sites after intravenous injection of ZnP2 NPs (50 µg/mL, 100 µL) in tumor-bearing mice. (b) In vivo fluorescence imaging within 48 h after intravenous injection of ZnP2 NPs (50 µg/mL, 100 µL) and drug tissue distribution in vivo after 24 h. (c) Tumor volume changes with time for various treatment groups. (d) Body weight changes with time for various treatment groups.

28


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Figure 7. H&E stained images of major organs (heart, liver, spleen, lung, and kidney) for different groups after 8 times treatment.

29



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Table 1. Spectroscopic parameters, singlet oxygen QYs (Φ∆) and half maximal inhibitory concentration (IC50) for P1, ZnP1 and ZnP2

ࣅ࢓ࢇ࢞ ࢇ࢈࢙ (nm)

IC50

ࣅ࢓ࢇ࢞ ࢋ࢓

Cpd

(nm)

Φ∆ (µg/mL)

In DCM

NPs in water

P1

426,521, 554, 598, 653

439,531, 569, 612, 662

664, 728

43%

7.8

ZnP1

432,563, 605

441,571, 612

609, 656

54%

5.7

ZnP2

459,610, 668

467,674

709, 760

79%

3.6

30


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ToC

31


Zinc(II) Metalated Porphyrins as Photothermogenic Photosensitizers

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