A Highly Efficient Red Metal-free Organic Phosphor for Time-resolved

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Biological and Medical Applications of Materials and Interfaces

A Highly Efficient Red Metal-free Organic Phosphor for Timeresolved Luminescence Imaging and Photodynamic Therapy Huifang Shi, Liang Zou, Kaiwei Huang, He Wang, Chen Sun, Shan Wang, Huili Ma, Yarong He, Jianpu Wang, Hai-Dong Yu, Wei Yao, Zhongfu An, Qiang Zhao, and Wei Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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ACS Applied Materials & Interfaces

A Highly Efficient Red Metal-free Organic Phosphor for Time-resolved Luminescence Imaging and Photodynamic Therapy Huifang Shi, †,‡,§ Liang Zou,⊥,§Kaiwei Huang, ‡ He Wang, ‡ Chen Sun, ‡Shan Wang, ‡ Huili Ma, ‡ Yarong He, ‡ Jianpu Wang, ‡ Haidong Yu, †,‡ Wei Yao, ‡ Zhongfu An,* †,‡ Qiang Zhao, *⊥ and Wei Huang*†,‡,⊥ †

Institute of Flexible Electronics (IFE), Northwestern Polytechnical University (NPU), 127 West

Youyi Road, Xi’an 710072, China. ‡

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

Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211800, China ⊥

Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for

Biosensors, Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommunications, Wenyuan Road 9, Nanjing 210023, China Keyword Organic phosphorescence, Organic semiconductor, Time-resolved luminescence imaging, Photodynamic therapy, Crystal engineering

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Abstract

Developing highly efficient red metal-free organic phosphors for biological applications is a formidable challenge. Here we report a novel molecular design principle to obtain red metal-free organic phosphors with a long emission lifetime (504.6 μs) and a high phosphorescence efficiency (14.6%) from the isolated molecules in crystal. Furthermore, the well-dispersed phosphorescent nanodots (PNDs) with the particle size around 5 nm are prepared through polymer-encapsulation in aqueous solution, which show good biocompatibility and low cytotoxicity. The metal-free PNDs are successfully applied to time-resolved luminescence imaging to eliminate background fluorescence interference in both vitro and vivo, as well as effective photodynamic anticancer therapy for the first time. This work will not only pave a pathway to develop highly efficient metalfree RTP materials, but also expand the scope of their applications to biomedical fields.

Introduction Room temperature phosphorescence (RTP) with rich excited state features in metal-free organic molecules has attracted considerable attention in various applications ranging from chemosensors15

and bioimaging6-9 to data encryption10-16 and anti-counterfeiting.17-19 Due to an inherent spin-

forbidden transition from the excited triplet state to the ground state and rapid non-radiative decay of triplet excitons via molecular motions or environment (oxygen, moisture, etc.) mediated quenching,20,21 however, organic phosphorescence can be largely obtained in an inert atmosphere or at low temperature.22-24 Recently, some feasible strategies have been proposed to design organic phosphorescent materials under ambient conditions through the following two pathways. One is introducing some organic units,25-28 like aromatic carbonyl groups, halogen, etc. to enhance the spin orbit coupling (SOC), further promoting intersystem crossing (ISC) of photo-induced excitons


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from singlet to triplet excited state.29,30 The other is constructing a rigid environment to restrain the non-radiative transition of triplet excitons through crystallization,31-34 host-guest doping methods,35-39 H-aggregation40-44 and so on.45-51 Despite great success in metal-free organic phosphorescent materials with colorful phosphorescent emission, it remains a formidable challenge to obtain highly efficient red phosphorescence under ambient condition due to intense non-radiative transitions of triplet excitons, such as self-quenching by π-π stacking, molecular rotations and variations, and inefficient ISC of excitons, thus limiting the potential applications of metal-free organic phosphorescence in biological fields to a great extent.

Scheme 1. Molecular design concept for highly efficient phosphorescence through both molecular (a) and crystal engineering (b). In view of the potential of red phosphorescence in biomedical applications, herein, we designed and synthesized a new benzothiadiazole (BT) derivative with highly efficient red phosphorescence, in which two bromine atoms were introduced into the BT unit to promote the ISC for RTP enhancement through heavy atom effect52 (Scheme 1a). Besides, two rigid and planar carbazole


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units were connected to BT chromophore for effectively suppressing non-radiative transition and intermolecular quenching caused by aggregations, such as triplet-triplet annihilation (TTA) between chromophores, via steric-hindrance effect in crystal (Scheme 1b). In addition, BT and carbazole units contained lone pair electron elements (S and N), which also facilitated the phosphorescence population. As expected, a metal-free red RTP molecule was achieved with a high efficiency of up to 14.6% and a long lifetime of 504.6 μs in solid state. Moreover, welldispersed organic phosphorescence nanodots (PNDs) were prepared by a top-down approach with ultrasmall particle size around 5 nm and long emission lifetime of 203.1 μs in aqueous media. Given the good biocompatibility and long-lived emission, time-resolved luminescence imaging in both vitro and vivo, as well as a photodynamic anti-cancer therapy were conducted initially by utilizing phosphorescent nanodots. Experimental Section Reagents and materials Unless other noted, all reagents used in the experiments were purchased from commercial sources without further purification. The reaction was carried out under a nitrogen atmosphere. Measurements 1

H NMR spectra were recorded on a Bruker Ultra Shield plus 400 MHz spectrometer with

tetramethylsilane (TMS) as the internal standard. Mass spectra were obtained on a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). UV-visible absorption

spectra

were

obtained

using

Shimadzu

UV-1750.

Steady-state

fluorescence/phosphorescence spectra and excitation spectra were measured using HitachiF-4600. Lifetimes were conducted on Edinburgh FLSP920 fluorescence spectrophotometer equipped with a microsecond flash-lamp (μF900). Photoluminescence quantum efficiency (PLQE) was collected


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on a Hamamatsu Absolute PL Quantum Yield Spectrometer C11347 under ambient condition. The fluorescence and phosphorescence quantum efficiencies were calculated through the integral proportion of each component in PL spectra. X-ray single crystal was achieved by using a Bruker SMART APEX-II CCD diffractometer with graphite monochromatic Mo-Kα radiation. Photos were taken by Cannon EOS 700D digital camera. Confocal luminescence imaging was carried out on an Olympus IX81 laser scanning confocal microscope (LSCM) equipped with a 40-immersion objective lens. Phosphorescence lifetime imaging was conducted on a DCS-120 confocal scanning fluorescence lifetime imaging microscopy (FLIM) systems. Synthesis of 4,7-dibromo-5,6-dicarbazolyl-2,1,3-benzothiadiazole (DBCz-BT) Dry N,N-Dimethylformamide (DMF, 15 mL) was injected to a 25 mL round-bottom flask charged with 9H-carbazole (0.50 g, 3.0 mmol) and potassium hydroxide (0.26 g, 4.5 mmol) under nitrogen protection. The resulting mixture was stirred at 45 oC for 2.5 h. Then a solution of 4,7-Dibromo5,6-difluoro-2,1,3-benzothiadiazole (0.32 g, 1.0 mmol) in dry DMF was slowly added into the flask and stirred for 3.0 h at 115 oC to obtain an orange precipitate. The raw product was collected and exacted with dichloromethane three times and recrystallized. The orange lamellar crystals were collected (0.11 g, 17.6%). 1H NMR (400 MHz, CDCl3): δ 7.67 (dd, J = 6.1, 2.7 Hz, 4H), 7.05-6.98 (m, 8H), 6.90 (dd, J = 6.1, 2.6 Hz, 4H). MALDI-TOF MS: 624.25; Anal. Calc. (%): C, 57.22; H, 2.088; N, 9.22; S, 4.695; Br, 26.777. Found (%): C, 57.61; H, 2.482; N, 8.97. S, 5.13. Results and Discussion The molecule 4,7-dibromo-5,6-dicarbazolyl-2,1,3-benzothiadiazole, namely DBCz-BT, was easily synthesized by one-step nucleophilic substitution (Scheme S2), whose chemical structure was fully characterized by NMR (Figure S1), matrix- assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS, Figure S2), elemental and single crystal analyses.


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Firstly, the photophysical properties of DBCz-BT in both solution and solid state were studied. A broad absorption band centered at 450 nm in dilute chloroform solution was observed (Figure S3), which was ascribed to intramolecular charge transfer. With the introduction of two bromine atoms in the molecule, the fluorescence intensity of the solution excited at 450 nm was very weak at 298 K, with a broad emission band at 605 nm (1.4 ns) in its PL spectrum (Figure S3 and S4). Interestingly, in solid state, its absorption band was red-shifted to 560 nm due to the intermolecular aggregation. To the best of our knowledge, this is the most red-shifted excitation wavelength of the reported red-emissive metal-free organic phosphors (Table S1), demonstrating its potential in biomedical fields. Surprisingly, the steady-state photoluminescence (PL) and phosphorescence spectra showed the similar profiles with a dominant emission band at around 602 nm under ambient conditions (Figure 1a and 1c). We speculated that the fluorescence of DBCz-BT in solid was almost quenched by the introduction of bromine atoms under ambient conditions. The red emission showed phosphorescence feature with a long lifetime of up to 504.6 μs (Figure 1b), which was further confirmed by temperature dependent PL spectra. With the decreased temperature from 300 to 60 K (Figure 1c), the steady-state PL intensity of DBCz-BT in solid state increased gradually, which confirmed that the red emission was assigned to phosphorescence rather than thermal activated delayed fluorescence (TADF).53-55 Moreover, the red phosphor can keep stable even under the oxygen atmosphere due to its compact crystal structure (Figure S5).


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Figure 1. Phosphorescence investigation of DBCz-BT powder under ambient conditions. (a) The excitation spectrum (black line) obtained by monitoring the emission at 602 nm and phosphorescence spectrum (red line) excited at 400 nm. (b) Lifetime decay profile of emission band at 602 nm. (c) Temperature dependent PL spectra excited at 375 nm with a filter of 450 nm; (d) Single crystal analysis of DBCz-BT with multiple intramolecular and intermolecular interactions; (e) Theoretical calculated spin-orbit coupling constant, energy diagram and spinorbital coupling (ξ) of DBCz-BT molecule. To understand the origin of phosphorescence, the molecular packing and interactions were analyzed by the X-ray single crystal study shown in Figure 1d. In a single molecule, it showed a non-planar molecular structure with multiple intramolecular interactions, including C-H∙∙∙π (2.880, 2.887, 2.903 and 2.930 Å), C-Br∙∙∙π (3.676 ~ 4.172 Å) and C-Br∙∙∙N (3.112 ~ 3.162 Å). Besides, there were four molecules around each core molecule with more intermolecular interactions, such


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as C-H∙∙∙π and C-Br∙∙∙π interactions, which restrained the non-radiative decay by limiting the molecular motions. Owing to the non-planar molecular configuration, no intermolecular π-π interactions were observed, which was beneficial to improve phosphorescent efficiency. Among those interactions, the heavy atom Br may play an important role in promoting ISC process for efficient RTP. To further verify our speculation, the theoretical calculations were conducted by density functional theory (DFT). As shown in Figure 1e, the SOC constants (ξ) of DBCz-BT between T1 and S0, as well as S1 and T1 were as large as 12.76 and 18.79 cm-1, respectively, due to the intramolecular heavy atom effect, which is larger than those of the reported metal-free organic phosphors.29 Notably, the SOC constants showed a small change with consideration of the intermolecular heavy atom interactions (Figure S6). These results indicated that the intramolecular heavy atom interactions played a dominant role in RTP enhancement, because the triplet excited state was mainly located on BT unit rather than carbazole (Figure 1e), which is further confirmed by the consistency of phosphorescence spectra between isolated DBFBT (the raw molecule without Cz shown in Figure 1a) in dilute solution at 77 K (Figure S7) and DBCz-BT in crystal. Therefore, we reasoned that the highly efficient phosphorescence of DBCzBT in crystal was from the isolated molecule in a rigid crystal environment owing to the special non-planar molecular structure, which is distinct from phosphorescence in the traditional metalfree organic RTP molecules in crystal.56,57


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Figure 2. Investigation of morphology and photoluminescence properties of phosphorescent nanodots (PNDs). (a) Photographs taken under room light and UV lamp and (b) HR-TEM image of PNDs dispersed in water. (c) PL spectrum of the PNDs (50 μg/mL) in aqueous solution. (d) Lifetime decay profile of emission band at 602 nm. To make this highly efficient metal-free organic phosphor be potential in the biological fields, a top-down method was used to prepare the organic phosphorescent nanomaterial to improve its biocompatibility. DBCz-BT was encapsulated by an amphiphilic copolymer, PEG-PPG-PEG (Pluronic F-127) to form uniform nanoparticles, which were well dispersed in aqueous solution. Under UV-lamp, it showed bright orange red emission (Figure 2a). From the high-resolution TEM image, it is found that the particle size reached as small as 5±2 nm (Figure 2b). Hence, we call this type of ultrasmall nanomaterials as phosphorescence nanodots (PNDs). As shown in Figure 2c, its PL spectrum revealed an intense emission with a dominated band at 602 nm excited at 405 nm under ambient condition, which is consistent with that in crystal state. Besides, it is found that the


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intensity of the emission at 602 nm is decreased with temperature increasing, indicating the emission at 602 nm is not from TADF (Figure S8). Furthermore, the lifetime of PNDs was as long as 203.1 μs (Figure 2d), demonstrating the phosphorescence feature.

Figure 3. Phosphorescence lifetime imaging in vitro and vivo. (a) Images of living HeLa cells with different delay times. (b) Images of a Zebra fish with different delay times. Note that HeLa cells were incubated with PNDs (75 μg/mL) and Hoechst 33342 (a cell nucleus dye, 1.0 μM) at 37 °C for 3 h. The excitation wavelength was 405 nm. PNDs (150 μg/mL) were injected into the brain of zebrafish. In a further set of experiment, we investigated the potential of PNDs for bio-applications. After HeLa cells incubated with PNDs (75 μg/mL) for 12 h, it is found that PNDs showed low cytotoxicity, which was confirmed by methylthiazolyltetrazolium (MTT) assay in Figure S9. Even in the high PND concentration of 200 μg/mL for 24 h, the cell viability was also over 85%. Next, taking advantage of the long emission lifetime of the PNDs to eliminate the background fluorescence in the living organs, a phosphorescence lifetime imaging microscopy (PLIM) in both live HeLa cells and zebrafish was carried out (Figure 3 and S10). As shown in Figure 3, both high quality long-lived signals were obtained in living HeLa cells and brain of zebrafish, which made


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it available to be recognized from the short-lived background fluorescence.58 To be specific, from the images with no time delay (Figure 3a), it was clearly observed that the signals were from the whole cells, the nuclear dye (Hoechst) showed strong interference. After 1 μs time delay, however, intense fluorescent signals from cell nucleus disappeared completely, leaving only long-lived phosphorescent signals of PNDs from the cytoplasm. Even after 10 μs, the phosphorescent signals were still visible. Moreover, the same technique was applied for in vivo imaging of a zebra fish, as shown in Figure 3b. Thus, the time-resolved luminescent technique was utilized to eliminate the short-lived fluorescence interference effectively by exerting a delay time. Therefore, this phosphorescent nanoprobe will be promising in real imaging in the complicated biological environment. Through Dexter energy transfer by TTA, the metal-free PNDs can also be utilized as a photosensitizer for photodynamic therapy (PDT). First, the HeLa cell was chosen as a model to study the anticancer effect of PNDs. Through control experiments, it was found that the luminescent signals were much weaker in the normal cells (3T3 and HL-7702 cells) than that in HeLa cells under the same condition (the same sample concentration and the incubation time), which suggested that our nanoprobe may label the cancer cells more easily (Figure S11 and S12). Subsequently, the ROS generation was studied in HeLa cells with incubation of both the PNDs (75 μg/mL) and 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA, 0.1 mL) that is a ROS tracker with green fluorescence in the presence of ROS. With the irradiation time prolonged from 0 to 8 min, the green fluorescent signals became brighter and brighter (Figure S13), indicating an efficient 1O2 generation under photo-excitation. The photo-generated 1O2 was expected to cause cell oxidative apoptosis and death to realize an anti-cancer therapy. Then PDT effect was systematically evaluated by MTT assay, confocal imaging and flow cytometry (FCM),


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respectively. As shown in Figure S9, the photo-induced cytotoxicity was obvious under a PDT process. At the concentration of 75 μg/mL, the cancer cell death ratio reached around 70%. With further increasing the concentration, the cell viability was less than 25%.

Figure 4. Photodynamic therapy application of phosphorescence nanodots. Confocal microscopy images of Calcein-AM- and PI-labelled HeLa cells, and flow cytometry quantification of annexin V-FITC- and PI-labeled HeLa cells (a) in dark control, (b) light control and (c) incubated with PNDs (75 μg/mL) under irradiation at 475 nm for 20 min. Cells were viewed in the green channel for Calcein-AM (λex = 488 nm, λem = 500-550 nm) and red channel for PI (λex =488 nm, λem = 600650 nm), respectively. To further verify the PDT capability of our phosphorescent nanodots, confocal imaging was carried out for HeLa cells labeled with calcein-AM, which is a live cell tracker with green


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fluorescence, and propidium iodide (PI), a death cell dye with red fluorescence, respectively. As shown in Figure 4a and 4b, in the blank dark and light control experiments, the HeLa cells cultivated without any treatment can keep alive after illumination for 0 or 20 min, which was also confirmed by the corresponding FCM results with the living cell ratios both over 90%. However, when HeLa cells were treated with the photosensitizer, PNDs (75 μg/mL) for 3 h, after 20 min irradiation and 3 h more cultivation, from the confocal imaging images (Figure 4c), we can clearly observe the cell morphologies changed greatly with large amounts of apoptotic and dead cells floating in the media, which were stained by PI, showing red fluorescence. The FCM measurement also quantified the PDT effect. After photodynamic procedure, the oxidation caused cancer cell apoptosis and death rate was over 80%. Thus, the metal-free organic phosphorescent nanodots would be promising as an anticancer agent in the photodynamic therapy. Moreover, we conducted a series of experiments for both photodynamic and photothermal effect of our phosphorescent nanoparticles in vitro. HeLa cells were also labelled with calcein-AM and PI, respectively. As shown in Figure S14, for the dark control groups incubated with PNDs, the cells lived healthily, indicating the good biocompatibility of our probes. For the HeLa cells incubated with both PNDs and N-acetyl-L-cysteine (NAC), an inhibitor of 1O2 generation, after 20 min irradiation under 475 Xe lamp and 3 h more cultivation, most of the cells kept alive normally, which suggested that there was less photothermal therapy (PTT) effect for this system. Moreover, for the cells only incubated with PNDs, after 20 min irradiation, most of the HeLa cells went to apoptosis and death, showing an efficient PDT effect.


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Figure 5. The relative tumor volume (a) and body weight (b) of mice in different groups with prolonged treated time; (c) H&E stained images of tumor slices from the tumor tissues of the control and treated groups. To further evaluate the therapy potential of the phosphorescent nanodots in the practical applications, we further conducted the photodynamic anti-cancer experiments in vivo. The PNDs (150 μg/mL) were injected subcutaneously into mice with HeLa cells and irradiated by Xe lamp at 475 nm for different periods of time (1, 8 and 15 days). As shown in Figure S15, in the blank control group without PNDs under the irradiation at 475 nm, the tumor part of mouse grew quickly with time prolonged. However, in the experimental group with subcutaneous injection of PNDs, the tumor parts of mice kept stable, which indicated an anti-cancer effect of our probe. Relative changes in the tumor volumes and body weight of tumor-bearing mice after treatment were shown in Figure 5a and 5b. Significant tumor suppression and shrinkage were observed from the animals with PDT process (DBCz-BT PNDs with irradiation). In all the control groups (control group 1:


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injection with PBS solution under irradiation at 475 nm; control group 2: injection with DBCz-BT PND solution without irradiation), the tumors grew rapidly at a comparable rate, over 5 times than the original size. While the body weight of the treated mice displayed no obvious loss, indicating the healthy states of all the mice. Moreover, the histological analysis to the tumor was performed via hematoxylin and eosin (H&E) staining to study the morphology (Figure 5c). H&E staining of the tumor sections gathered from various treatment groups at the end of subcutaneous injection treatment at low magnification. Compared to the control groups from which densely packed neoplastic cells are observed throughout the mice, the tumors treated with PDT show a drastically impacted tumor architecture and significantly reduced cell density, indicating the cell apoptosis and death of tumor tissue caused by PDT process. Conclusion In summary, we have developed a new red emissive metal-free organic phosphor DBCz-BT with an emission lifetime of 504.6 μs and a high quantum yield of 14.6 % in solid state under ambient conditions. Based on both experimental data and theoretical simulation, we speculated that the highly efficient red phosphorescence stemmed from isolated molecule with intramolecular heavy atom effect in rigid crystal environment for the first time. Furthermore, well-dispersed organic phosphorescence nanodots were prepared by a top-down approach with ultrasmall particle sizes around 5 nm and a long emission lifetime of 203.1 μs in aqueous media. Given the good biocompatibility and long-lived emission lifetime, a time-resolved luminescence imaging in both vitro and vivo was applied to eliminate the background fluorescence interference effectively, which was the first example to use time-resolved technique in a metal-free organic phosphorescent system so far as we know. This study not only provides an effective molecular design strategy to achieve highly efficient metal-free organic phosphorescence materials, but also explores their


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potential in biomedical fields, such as time-resolved luminescence imaging and photodynamic cancer therapy, although with relative low tissue penetration due to the excitation limitation. In the future, it will offer a guideline to design more efficient purely organic phosphorescence materials with red-shifted excitation and emission for deeper therapy study. Supporting Information Experimental section, photophysical study, calculated data, additional cell experiments and animal study. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *E-mail: [email protected]. * E-mail: [email protected] *E-mail: [email protected]. Author Contributions These authors contributed equally.

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Notes The authors declare no competing financial interest. Acknowledgment This work is supported by the National Natural Science Foundation of China (21875104, 51673095, 21507098 and 91833304), National Basic Research Program of China (973 Program, No. 2015CB932200), Natural Science Fund for Distinguished Young Scholars of Jiangsu Province (BK20180037), Primary Research & Development Plan of Jiangsu Province (BE2016770) and the


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Natural Science Fund for Colleges and Universities (17KJB430020) of Jiangsu Province. We are grateful to the High-Performance Computing Center of Nanjing Tech University for supporting the computational resources. Huifang Shi and Liang Zou contributed equally to this work. References 1 Zhang, K.; Yu, Q.; Wei, H.; Liu, S.; Zhao, Q.; Huang, W. Long-Lived Emissive Probes for Time-Resolved Photoluminescence Bioimaging and Biosensing. Chem. Rev. 2018, 118, 17701839. 2 Yu, Y.; Kwon, M. S.; Jung, J.; Zeng, Y.; Kim, M.; Chung, K.; Gierschner,J.; Youk, J. H.; Borisov, S. M.; Kim, J. Room-Temperature-Phosphorescence-Based Dissolved Oxygen Detection by Core-Shell Polymer Nanoparticles Containing Metal-Free Organic Phosphors. Angew. Chem. Int. Ed. 2017, 56, 16207-16211. 3 Lehner, P.; Staudinger, C.; Borisov, S. M.; Klimant, I. Ultra-Sensitive Optical Oxygen Sensors for Characterization of Nearly Anoxic Systems. Nat. Commun. 2014, 5, 4460. 4 Fermi, A.; Bergamini, G.; Roy, M.; Gingras, M.; Ceroni, P. Turn-on Phosphorescence by Metal Coordination to a Multivalent Terpyridine Ligand: A New Paradigm for Luminescent Sensors. J. Am. Chem. Soc. 2014, 136, 6395-6400. 5 Wu, Q; Ma, H; Ling, K; Gan, N; Cheng, Z; Gu, L; Cai, S; An, Z; Shi, H; Wang, H. Reversible Ultralong Organic Phosphorescence for Visual and Selective Chloroform Detection. ACS Appl. Mater. Interfaces 2018, 10, 33730-33736.


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A Highly Efficient Red Metal-free Organic Phosphor for Time-resolved

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