Hyperbranched Phosphorescent Conjugated Polymer Dots with

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Hyper-Branched Phosphorescent Conjugated Polymer Dots with Iridium(III) Complex as the Core for Hypoxia Imaging and Photodynamic Therapy Zhiying Feng, Peng Tao, Liang Zou, Pengli Gao, Yuan Liu, Xing Liu, Hua Wang, Shujuan Liu, Qingchen Dong, Jie Li, Bingshe Xu, Wei Huang, Raymond Wai Yeung Wong, and Qiang Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09721 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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ACS Applied Materials & Interfaces

Hyper-Branched Phosphorescent Conjugated Polymer Dots with Iridium(III) Complex as the Core for Hypoxia Imaging and Photodynamic Therapy Zhiying Feng,a,b,† Peng Tao,a,b,† Liang Zou,b Pengli Gao,b Yuan Liu,a Xing Liu,a Hua Wang,a,* Shujuan Liu,b Qingchen Dong,a Jie Li,a Bingshe Xu,a Wei Huang,b Wai-Yeung Wong,c and Qiang Zhaob,* a

Research Center of Advanced Materials Science and Technology and MOE Key Laboratory of

Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Taiyuan 030024, P. R. China. E-mail: [email protected] b

Key Laboratory for Organic Electronics and Information Displays and Institute of Advanced

Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications (NUPT), Nanjing 210023, P. R. China. E-mail: [email protected] c

Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic

University, Hung Hom, Kowloon, Hong Kong, P. R. China. † Z. Y. Feng and P. Tao contributed equally to this work. KEYWORDS: hyper-branched polymer dots, hypoxia imaging, iridium(III) complexes, phosphorescence, photodynamic therapy

ACS Paragon Plus Environment

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ABSTRACT

Real-time monitoring the contents of molecular oxygen (O2) in tumor cells is of great significance in early diagnosis of cancer. At the same time, the photodynamic therapy (PDT) could be realized by highly toxic singlet oxygen (1O2) generated in situ during the O2 sensing, making it one of the most promising methods for cancer therapy. Herein, the iridium(III) complex cored hyper-branched phosphorescent conjugated polymer dots with the negative charges for hypoxia imaging and highly efficient PDT was rationally designed and synthesized. The incomplete energy transfer between the polyfluorene and the iridium(III) complexes realized the ratiometric sensing of O2 for the accurate measurements. Furthermore, the O2-dependent emission lifetimes are also used in photoluminescence lifetime imaging and time-gated luminescence imaging for eliminating the auto-fluorescence remarkably to enhance the signal-to-noise ratio of imaging. Notably, the polymer dots designed could generate the 1O2 effectively in aqueous solution, and the image-guided PDT of the cancer cells was successfully realized and investigated in detail by confocal laser scanning microscope. To the best of our knowledge, this represents the first example of the iridium(III) complex cored hyper-branched conjugated polymer dots with the negative charges for both hypoxia imaging and PDT of cancer cells simultaneously.

1. Introduction Very recently, photodynamic therapy (PDT), in which tumor cells could be killed by light-induced generation of the singlet oxygen (1O2), has been considered as one of the most promising candidates in the therapy of cancer owing to its highly efficient therapeutic effect, controllable and high selectivity for the lesion area, and low damage to healthy tissues. 1-5


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Molecular oxygen (O2) plays significant roles in various important physiological processes, such as signaling pathway, aerobic respiration, oxidative phosphorylation, and numerous enzymatic transformations.6-8 In general, the content of O2 in tumor is much lower than that of the healthy tissues due to the excessive consumption of O2 for the rapid proliferation of tumor, that is, the tumor is under the oxygen deprivation (hypoxia).9-11 Naturally, the O2 could be regarded as a unique informative indicator for identifying the potential tumor tissue in the early-stage diagnosis of cancers. Furthermore, the O2 could be transformed to the reactive 1O2 which is quite toxic to the cancer cells. Thus, mapping of O2 contents in the living cancer cells and then killing the cancer cells by light-induced generation of the 1O2 are of great significance in the cancer diagnosis and therapy.12-14 Up to date, there are many techniques for hypoxia imaging, such as electron paramagnetic resonance, magnetic resonance imaging, pulse oximetry, and positron emission tomography.15-18 Compared with these techniques, optical imaging techniques have received much attention in the detection of intracellular O2.19-20 Among the various O2-responsive dyes suitable for the hypoxia imaging, phosphorescent transition-metal complexes (PTMCs) have special merits owing to the easy phosphorescence quenching by efficient and reversible energy transfer from the excited triplet states of PTMCs to the ground states of O2, giving rise to 1O2 simultaneously.21 Moreover, PTMCs show excellent photostability, large Stokes shifts, and relatively long emission lifetimes beneficial for biosensing and bioimaging.22-28In addition, the signal-to-noise ratios in the sensing of intracellular O2 can be largely improved by eliminating the auto-fluorescence interference owing to the long phosphorescence lifetime of PTMCs via time-resolved luminescence imaging techniques.29


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Many efforts have been devoted in the design and construction of the agents for the highly efficient image-guided PDT.30-33 The agents based on small molecules reported often suffer from high cytotoxicity, low molar absorption coefficient in the visible region, and poor solubility in water. And the majority of O2 probes are the single intensity-based sensing, leading to the inaccurate measurement caused by external influence. So these drawbacks of small molecular agents limit the practical applications. To solve these problems, conjugated polymer dots involving PTMCs were regarded as a promising candidate for the simultaneous hypoxia imaging and image-guided PDT owing to their attractive properties, for instance, excellent light-harvesting capability, good solubility in water and light-amplifying properties.34-37 Recently, we reported a series of PTMC-containing polymer dots for PDT. In these polymer dots, the iridium(III) or platinum(II) complexes were introduced into the polyfluorene to serve as oxygen sensing moiety with the positively charged quaternary ammonium salts chemically bonded to the fluorene segments to guarantee the solubility in aqueous solution.38-40 These polymer dots with the positive charges on the surface can indeed realize the excellent water-solubility and easily enter into the cells through the surface of the cell membrane. However, these polymer dots with positive charges could not be the ideal candidates for the sensing and imaging in vivo, because there exist various biomacromolecules with the negative charges, such as biological proteins and enzymes in the blood of the living tissues, and these negatively charged biomacromolecules may potentially interact with the positively charged polymer dots by the electrostatic attractions, leading to the aggregation and accumulation of the polymer dots in vivo.1, 37, 41 Thus, the rational design and construction of the negatively charged polymer dots to overcome the above-mentioned drawbacks are quite urgent. From the perspective of the molecular design, the combination of the iridium(III) complex cored hyper-branched conjugated polymer and the


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negative charges transformable polymer poly(styrene-co-maleic anhydride) (PSMA) could be a good solution to solve this problem. Different from the previously reported polymers with the PTMC moieties in the linear chains, which often suffer from the strong interchain interactions leading to the self-quenching of the phosphorescence,38,

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the iridium(III) complex cored

hyper-branched conjugated polymer possesses the compact three-dimensional branched topological structures and the iridium(III) complexes are well protected by the branched structures, avoiding the self-quenching of the phosphorescence effectively.43-46 In addition, by incorporating the negatively charged PSMA into the surface of the constructed polymer dots, the PSMA could form a polymer shell to prevent the polymer dots from potentially interacting with solvents and other interfering molecules, especially in the environment of living cells. So, the incorporation of PSMA could improve the stability and anti-interference ability under the complicated conditions. In this contribution, phosphorescent conjugated polymer dots with multifunction for ratiometric hypoxia imaging and photodynamic therapy of cancer cells were rationally designed and constructed. As illustrated in Figure 1, we selected the oxygen-sensitive red phosphorescent fac-tris(1-phenylisoquinolinato-N,C2')iridium(III) as the core and the oxygen-insensitive blue fluorescent 9,9-dioctylfluorene as the conjugated backbones of the hyper-branched conjugated polymer. By the methods of coprecipitation, the PSMA was successfully incorporated into the phosphorescent conjugated polymer dots to provide the negative charges (~-25.86 mV of the zeta potential) and ensure the water-solubility and biocompatibility. The resulting iridium(III) complex cored hyper-branched conjugated polymer dots show the quite small size (~20 nm) formed by the hydrophobic interactions and exhibit excellent oxygen sensing performance owing to the effective energy transfer from the excited triplet states of iridium(III) complexes to the


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ground states of O2. The incomplete Förster resonance energy transfer (FRET) between the polyfluorene segments and the cored iridium(III) complexes realize the ratiometric sensing of O2 for the accurate measurements independent on the concentrations of the probe and the interferences existent in the sensing system. Furthermore, the relatively long emission lifetimes of the phosphorescence were also very sensitive to the O2. This O2-dependent emission lifetimes could be used in photoluminescence lifetime imaging microscopy (PLIM) and time-gated luminescence imaging (TGLI) for eliminating the background of auto-fluorescence with short-lived lifetimes remarkably to enhance the reliability of imaging. More notably, the polymer dots designed could generate the 1O2 effectively, and the image-guided PDT of the cancer cells was successfully realized and investigated in detail by confocal laser scanning microscope (CLSM). To the best of our knowledge, this is the first example of the iridium(III) complex cored hyper-branched conjugated polymer dots with the negative charges for both hypoxia imaging and PDT of cancer cells. 2. Results and Discussion 2.1. Design, Synthesis, Construction and Characterizations The synthetic route of hyper-branched phosphorescent conjugated polymer Ir-HPC was shown in

Scheme

1.

The

monomers

2,2'-(9,9-dioctyl-9H-fluorene-2,7-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane) fac-tris(1-(4-bromophenyl)isoquinolinato-N,C2')iridium(III)

(M2)

(M1), and

2,7-dibromo-9,9-dioctyl-9H-fluorene (M3) were synthesized by the previous reports.47 The target polymer was accomplished through highly efficient Suzuki polymerization reaction, and the polymer

was

capped

by

2-(9,9-dioctyl-9H-fluoren-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

adding and


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2-bromo-9,9-dioctyl-9H-fluorene to couple the terminal aromatic bromide or borate ester. The polymer was precipitated in methanol, and then extracted by Soxhlet extraction, finally, purified by column chromatography to obtain a solid powder. The polymer was characterized by 1H NMR,

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C{1H} NMR and GPC. The actual contents iridium(III) complexes in hyper-branched

conjugated polymer was slightly higher than that of the feed ratios probably caused by the extra formation of linear polyfluorenes due to the different reaction activity between iridium(III) complex and bromofluorene. The weight-average molecular weights (Mw) of the Ir-HPC was estimated by GPC to be 21100 and a polydispersity index (PDI) of 1.74 as shown in Table S1. We note that the Mw of Ir-HPC is lower than those in the previously reported polymers,35, 48 which could be the result of the iridium(III) complex as the core of polymer and much higher feed ratios of the iridium(III) complex in the formation of this hyper-branched conjugated polymer. The PSMA was incorporated for the construction of phosphorescent conjugated polymer dots (abbreviated as Ir-HPC/PSMA dots) to increase the water-solubility, biocompatibility and reduce the diameters of the resulting polymer dots. The Ir-HPC/PSMA dots were prepared by coprecipitation illustrated in Figure S1. As shown in Figure 2a, the morphology of the Ir-HPC/PSMA dots in aqueous solution was observed by transmission electron microscope (TEM) and the size of the dots was determined to be ~17 nm, which was formed and controlled by the hydrophobic interactions. The diameter distributions of the polymer dots were further characterized by dynamic light scattering (DLS) shown in Figure 2b. It could be found that the average diameter was estimated to be ~22 nm, which is slightly larger than that obtained using TEM method consistent with the previously studies.49 The zeta potential of the Ir-HPC/PSMA dots was ~-25.86 mV (Figure 2c), for comparison, the PSMA-free Ir-HPC dots was also


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prepared and the zeta potential was ~-2.03 mV, clearly suggesting that the PSMA was successfully incorporated into the polymer dots. Owing to the negative charge on the surface of the particle, the resulting Ir-HPC/PSMA dots dispersions are clear and exhibit quite stability at least for one month without aggregation (Figure 2d), indicating that these phosphorescent polymer dots have great potential applications in the biomedical field. 2.2. Photophysical Properties The photophysical properties of polymer Ir-HPC and Ir-HPC/PSMA dots were investigated and shown in Figure 2e, 2f. Both Ir-HPC in THF and Ir-HPC/PSMA dots in aqueous solution show intensive absorption in the region of 300-440 nm, which is corresponding to π-π* transition absorption of fluorene moieties.47,

50-51

The absorption of PSMA and PSMA dots was also

measured for comparison (Figure S2). Slightly different from Ir-HPC in THF, the Ir-HPC/PSMA dots exhibit shoulder peak at 430 nm, indicating the formation of β-phase of polyfluorene in this polymer dots.50 The absorption band of the cored iridium(III) complex almost could not observed due to the quite low iridium(III) complex content in the polymer. However, the emission spectra of Ir-HPC in THF and Ir-HPC/PSMA dots in aqueous solution are quite different under air atmosphere. The α-phase is a disordered glassy phase of polyfluorene, where the molecular chains exist in random orientations, while the β-phase is more ordered and flat in conformation.50-51 The Ir-HPC in THF exhibits intensive blue emission with the emission peak at 417, 439 nm originated from emitting of α-phase polyfluorene, but nearly no emission in red region, which should be the result of relatively low FRET efficiency from polyfluorene segments to the cored iridium(III) complexes in the free states of the polymer and the phosphorescence quenching caused further by molecular oxygen dissolved in THF under air atmosphere. In contrast to Ir-HPC in THF, the Ir-HPC/PSMA dots in aqueous solution exhibit double emission


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band, one located at 437 nm with shoulder peak at 463 nm, another located at 630 nm. The former blue emission band could be attributed to the emission of β-phase of polyfluorene segments,50 indicating that the polyfluorene chains in Ir-HPC/PSMA dots stack orderly with rather smaller inter-chain distance. The later red emission band is then corresponding to emission of Ir(III) complex owing to the enhanced inter-chain FRET from polyfluorene chains to the cored Ir(III) complex in the polymer dots, as illustrated in our previously reports.50-51 It is interesting to note that the PL spectrum in the blue region of Ir-HPC/PSMA dots is much narrower and sharper (λFWHM = 12 nm vs 45 nm) than that of polymer Ir-HPC in THF, which is consistent with the typical luminous properties of nanoparticles.52-55 Moreover, the absolute solution quantum yield of the Ir-HPC/PSMA dots for the total emission was estimated to be approximately 0.22 under the air atmosphere, which is much higher than that of the corresponding linear polymers.38 2.3. Oxygen Sensing of Ir-HPC/PSMA Dots in Aqueous Solution Oxygen sensing performance of Ir-HPC/PSMA dots in aqueous solution was further investigated. As depicted in Figure 3a, with increasing O2 contents from 0% to 100%, the intensity of oxygen-insensitive emission peak at 470 nm was almost constant, while the intensity of emission peak at 630 nm corresponding to iridium(III) complex was very sensitive to O2 contents and gradually weakened by efficient FRET from the excited triplet states of iridium(III) complex to the ground states of molecular oxygen (3O2), which gives rise to a perfect ratiometric probe for the O2 sensing. The wavelength gap between the blue and red emission peaks (reaching 160 nm) was large enough to ensure the accurate measurements of the intensities of two emissions to give the ratiometric value. Moreover, the red emission band of Ir-HPC/PSMA dots in aqueous solution increase remarkably upon decreasing the O2 contents, resulting in an obvious alteration of emission color (from blue to purple), which could also be realized by the naked eyes


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detection. At the same time, although the lifetime of the blue emission did not change and remained 1.2 ns under different O2 contents (Figure S5), the lifetime of the red emission band was very sensitive to O2 contents and decreased from 1.1 μs to 0.45 μs when the O2 contents was increased from 0% to 100% (Figure 3b and Table S2). Thus, the property of emission lifetimes sensitive to oxygen could also be used to realize the O2 sensing. In order to quantitatively determine the contents of O2 in aqueous solutions, the ratios of the intensity of phosphorescence at 630 nm to the fluorescence at 470 nm were defined as RI0 = Ip0/If0 and RI = Ip/If in the absence and presence of O2, respectively. The emission lifetimes at 630 nm were defined as τ0 and τ in the absence and presence of O2, respectively. The KSV value was determined by the Stern-Volmer equation: 𝑅I0 𝜏0 = = 1 + 𝐾SV [O2 ] 𝑅I 𝜏 where [O2] was the contents of O2.39, 56 As shown in Figure 3c, 3d and S6, a linear relation between RI0/RI and [O2], τ0/τ and [O2] has been obtained. The quenching constant KSV was calculated to be 2.42 (3.18×10-3 mmHg-1). Moreover, we also measured the emission lifetimes at 630 nm under various contents of O2. The lifetimes of phosphorescence also exhibit a linear relationship with the contents of O2 as shown in Figure 3d and the KSV of 2.17 (2.86×10-3 mmHg-1) can be obtained. The lifetime-based KSV value was in consistent with that based on the intensity of emission. The excellent linearity implied the good reliability and sensitivity of Ir-HPC/PSMA dots for oxygen quantification. The photostability is an important factor of the probe. As shown in Figure S3, we evaluated the photostability of the Ir-HPC/PSMA dots in aqueous solution under various conditions. After continuous illumination at 370 nm even for 30 min, both two emission bands of the polymer dots did not show any detectable intensity loss, indicating that the Ir-HPC/PSMA dots have excellent


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stability to resist the photobleaching. In addition, the spectrum stability is another consideration for the excellent probe. Thus, we further examined the potential effect of pH variations on the spectrum, as shown in Figure S4, under the air condition, the ratios of red and blue intensity are nearly inependent on the pH values from 4.5-9.0, indicating that the nanoprobe constructed could not be interfered by the complicated environment. 2.4. Ratiometric Luminescence Hypoxia Imaging of Ir-HPC/PSMA Dots in Living Cells The hypoxia imaging performance of Ir-HPC/PSMA dots was evaluated in HepG2 cells. Under the content of 21% O2 and 5% CO2, HepG2 cells were cultured at 37 °C for 24 h. The Ir-HPC/PSMA dots were added to the medium at a concentration of 8 μg·mL–1 and then cells were incubated for another 2 h under the O2 contents of 21% and 2.5%, respectively. The wavelength of 405 nm was chosen as the excitation wavelength for hypoxia imaging. The images were depicted in Figure 4a, the overlay of the blue and red confocal luminescence demonstrated that the Ir-HPC/PSMA dots were located in the cytoplasm of the living cells. Upon decreasing the O2 content from 21% to 2.5%, the red emission intensity of iridium(III) complex collected at 600-700 nm shows the remarkable enhancement owing to the nature of phosphorescence emission. Compared with the red emission, the intensity of blue emission from polyfluorene moieties at 420-490 nm remained nearly unchanged under various contents of O2. Moreover, by calculating the ratio of intensity of emission at 600-700 nm and that at 420-490 nm, the actual O2 contents in the living cells could be obtained. When the living HepG2 cells were cultured at different O2 contents, as depicted in Figure 4b, the value of emission ratio changed evidently, demonstrating that the excellent ratiometric O2 sensing properties of the Ir-HPC/PSMA dots suitable for the hypoxia imaging in living cells. 2.5. Photoluminescence Lifetime Imaging of Ir-HPC/PSMA Dots in Living Cells


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Considering the long phosphorescence lifetime property of the Ir-HPC/PSMA dots, the signal-to-noise ratios in the sensing of intracellular O2 could be evidently improved by eliminating the auto-fluorescence interference via time-resolved luminescence imaging techniques.29 Then, we conducted the photoluminescence lifetime imaging of HepG2 cells under the O2 contents of 21% and 2.5%, respectively. Analogous to that in aqueous solution, as shown in Figure 4c, the cells cultured under 2.5% O2 exhibited much longer lifetime. And the lifetimes of red emission under 21% O2 and 2.5% O2 were estimated to be 172 and 215 ns in HepG2 cells, respectively. To highlight the advantage of the long-lived phosphorescence of Ir-HPC/PSMA dots, time-gated luminescence imaging was utilized to map the intracellular O2, and the emission intensity-based images in various time intervals have been shown in Figure 4c. By means of analyzing the signals in different lifetime intervals of 0-1000 ns and 180-1000 ns, and under various O2 contents, the images of cells under 2.5% O2 were brighter than that of cells under 21% O2, confirming that Ir-HPC/PSMA dots as a long-lived intracellular O2 probe was extremely effective for eliminating the auto-fluorescence background with short-lived lifetimes. In view of these results obtained, a time-resolved fluorescent/phosphorescent and ratiometric nanoprobe platform with negative charges has been constructed and demonstrated successfully, which realizes the detection of intracellular O2 quantitatively and gives more sensitive and reliable measurements in the complicated environment by application of PLIM and TGLI techniques. 2.6. Photodynamic Therapy of Ir-HPC/PSMA Dots in Living Cells In the light of the excellent performance of hypoxia imaging in living cells, we further made full use of the phosphorescence nature of nanoprobe. The highly active singlet oxygen could be generated in situ in the process of the oxygen detection. First, in order to evaluate the 1O2 generation ability of polymer dots, the experiment to determine the singlet oxygen quantum


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yields was carried out. 2,2'-(Anthracene-9,10-diylbis(methylene))dimalonic acid (ABDA) was selected as an 1O2 indicator and tris(2,2'-bipyridine)dichlororuthenium(II) (Ru(II)(bpy)3Cl2) (ΦΔ = 0.41 in water) was used as a reference to calculate the 1O2 quantum yield of Ir-HPC/PSMA dots.38,

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Owing to the relatively strong absorbance in 430 nm induced by the β-phase of

polyfluorene formed in this polymer dots, the blue light at 420 nm was used as the light source of PDT. As illustrated in Figure 5, the absorption band (341, 359, 378 and 399 nm) of the mixtures of photosensitizer (Ir-HPC/PSMA dots or Ru(II)(bpy)3Cl2) and ABDA decreased gradually because of the decomposition of ABDA by the 1O2 formed under the irradiation of 420 nm. The 1

O2 quantum yield of Ir-HPC/PSMA dots was determined to be as high as 0.52. The high

light-induced efficiency of 1O2 for Ir-HPC/PSMA dots is beneficial to PDT in living cells, because the singlet oxygen could give rise to reactive oxygen species (ROS) and thereby induce apoptosis and death of the cell.58 Under the irradiation of light, 2,7-dichlorifluoresceindiacetate (DCFH-DA) was served as an indicator to trace the singlet oxygen generation of Ir-HPC/PSMA dots in living cells, because it could be converted into DCFH in living cells, which is further oxidized by ROS to emissive 2,7-dichlorofluorescein (DCF).40 The production of singlet oxygen monitored by DCFH in aqueous solution was carried out and investigated initially. Exposure of the mixtures of DCFH and phosphorescent Ir-HPC/PSMA dots under the light at 420 nm made the fluorescence turn on at 521 nm (Figure S7), confirming that the DCFH could be used to monitor the presence of ROS in aqueous solution. The fact that fluorescence alteration could not be observed in the controlled experiment indicated that the generation of 1O2 was indeed caused by Ir-HPC/PSMA dots. Next, we investigated and confirmed the generation of the intracellular 1

O2. After cells were coincubated with DCFH-DA and Ir-HPC/PSMA dots, under different

irradiation conditions, two emission channels of 505-565 (the emission from DCF) and 420-490


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nm (the emission from Ir-HPC/PSMA dots) were collected, respectively. Emission intensity from channel of 505-565 nm was remarkably enhanced after exposure of 420 nm for 10 min (Figure 6a). Different from the former, the intensity of emission at 420-490 nm remained unchanged after exposure. Thus, in living cells, the ROS generation was also proved to be induced by the Ir-HPC/PSMA dots under irradiation at 420 nm, which is further confirmed by the controlled experiment (Figure S8). Based on the above results, the image-guided photodynamic therapy based on the constructed Ir-HPC/PSMA dots was further explored in HepG2 cells with Annexin V-FITC/propidium iodide (PI) employed to trace and differentiate the stages of apoptosis and death of the cell. 40, 56 The progress of cell apoptosis induced by irradiation was also monitored by the real-time fluorescence imaging in detail. Under 21% O2 atmosphere, HepG2 cells were cultured with Ir-HPC/PSMA dots at 37 °C for 2 h and then exposed under a 420 nm lamp with the power of 25 mW·cm-2. After the cells were stained with Annexin V-FITC/PI, the changes of the cell irradiated were observed in real-time by means of CLSM. We can see from Figure 6b, highly efficient PDT effect of Ir-HPC/PSMA dots to kill cancer cells was achieved, which was also confirmed by adding the ROS scavenger Vitamin C (VC) in the controlled experiment. Moreover, as shown in Figure 7, in the process of in situ observation of cell death induced by PDT, after exposure under 420 nm light, Annexin V-FITC stained in cell membrane showed the increasing intensity of green fluorescence. However, we could not observe the red fluorescence from PI. The cells were further incubated in the dark. After 2 h, as shown in Figure 7, the red fluorescence of PI could also be observed, demonstrating that the necrosis process of the cells occurred and should be attributed to the ROS generated in Ir-HPC/PSMA dots-based PDT. In contrast, in the controlled experiments, the cell death was not observed even after 11 h (Figure S9-S14).


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Accordingly, the generated ROS of Ir-HPC/PSMA dots-based PDT was responsible for the apoptosis and death of the cell. The MTT assay was also carried out in HepG2 cells to further confirm the efficient performance of photodynamic therapy (Figure S15). The cell viability was decreased quite quickly upon increasing the dose of polymer dots and exposure time, confirming the highly efficient PDT of Ir-HPC/PSMA dots for killing cancer cells. The MTT assay was also in agreement with the real-time luminescence imaging. 3. Conclusions In summary, the first example of iridium(III) complex cored hyper-branched phosphorescent conjugated polymer dots with the negative charges for hypoxia imaging and highly efficient photodynamic therapy was rationally designed and synthesized. The oxygen-sensitive red phosphorescent iridium(III) complex was selected as the core and the oxygen-insensitive blue fluorescent 9,9-dioctylfluorene was served as the conjugated backbones of the hyper-branched conjugated polymer. The PSMA was incorporated into the phosphorescent conjugated polymer dots by coprecipitation to provide the negative charges and ensure the water-solubility and biocompatibility. The intracellular O2 content was investigated using this nanoprobe by ratiometric imaging, photoluminescence lifetime imaging and time-gated luminescence imaging. The detection accuracy could be enhanced by the ratiometric imaging, the PLIM and TGLI realized by this novel phosphorescent polymer dots could effectively eliminate the auto-fluorescence interference to provide more reliable and sensitive measurements. Owing to the phosphorescent nature and high singlet oxygen quantum yield of nanoprobe attributed to the incorporation of iridium(III) complex, the potential applications in image-guided PDT were confirmed by real-time luminescence imaging and MTT assay of light-induced apoptosis and death of cell in situ. These preliminary results demonstrated that the new phosphorescent


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polymer dots-based diagnosis-therapy platform would be the promising candidates for the future cancer therapy. 4. Experimental 4.1. Experimental Information The details of materials, instruments and methods, cell culture and imaging and so on can be found in Supporting Information. 4.2. Synthetic Section The Synthesis of Iridium(III) Complex Cored Hyper-Branched Phosphorescent Conjugated Polymer (Ir-HPC). The monomers (M1, M2 and M3) were synthesized by the previous reports.47 The compounds fac-tris(1-(4-bromophenyl)isoquinolinato-N,C2')iridium(III) (M2) (30 mg, 0.029 mmol), 2,2'-(9,9-dioctyl-9H-fluorene-2,7-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane) (M1) (470 mg, 0.731 mmol), 2,7-dibromo-9,9-dioctyl-9H-fluorene (M3) (380 mg, 0.690 mmol) together with Pd(PPh3)4 (2 mol%), methyl trioctyl ammonium chloride, K2CO3 aqueous solution (2 mol/L) were dissolved in toluene (20 mL), and then stirred under 100 oC with N2 protection for

48

h.

Subsequently,

the

polymer

was

capped

2-(9,9-dioctyl-9H-fluoren-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

(20

by mg)

adding with

continuous stirring for 12 h, and then 2-bromo-9,9-dioctyl-9H-fluorene (20 mg) was added and the reaction was continued for another 12 h. After the reaction was completed and cooled to room temperature. Under continuous stirring, the reaction solution was poured into methanol. The precipitated polymer was collected by filtration and then purified with acetone by Soxhlet extraction to remove the small molecules. Finally, the crude product was further purified by column chromatography (silica gel; CH2Cl2: THF = 10:1) to obtain the solid powder (65% yield). 1

H NMR (400 MHz, CDCl3): δ (ppm) 9.02 (br, Ar-H of Ir complex), 8.32 (br, Ar-H of Ir


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complex), 7.99 (d, Ar-H of Ir complex), 7.86-7.34 (m, Ar-H of fluorene, Ir complex), 6.88-6.84 (m, Ar-H of fluorene, Ir complex), 2.13 (br, CH2(CH2)6CH3 of fluorene,), 1.26-1.15 (m, CH2(CH2)6CH3 of fluorene), 0.82 (t, CH2(CH2)6CH3 of fluorene).

13

C{1H} NMR (100 MHz,

CDCl3): δ (ppm) 151.84, 140.52, 140.05, 128.81, 127.24, 126.18, 121.52, 120.00, 55.37, 40.42, 31.83, 30.07, 29.26, 23.94, 22.64, 14.10, 1.05. The Preparation of Hyper-Branched Phosphorescent Conjugated Polymer Dots (Ir-HPC/PSMA dots). The Ir-HPC/PSMA dots were prepared by the method of reprecipitation as illustrated in Figure S1. Firstly, the hyper-branched polymer Ir-HPC and PSMA were dissolved separately into tetrahydrofuran (THF) with concentration of 1 mg/mL. Secondly, the two solutions were mixed with the content ratio between Ir-HPC to PSMA of 5:2. The mixture solution was diluted with THF, which acquire the Ir-HPC concentration of 40 μg/mL and PSMA concentration of 16 μg/mL. The diluted mixtures solution was agitated to form homogeneous solutions. Thirdly, the 5 mL of the mixture solution was injected quickly into 10 mL of deionized water while sonicating solution (the power of sonication is 110 W and the temperature is 38 oC). The THF was removed by nitrogen stripping, and the volume of the resulting solution was concentrated to 5 mL, followed by filtration through a 0.22 micron filter.

Associated Content Supporting Information The materials of detailed description about testing and cell experiments is available free of charge via the Internet at http://pubs.acs.org.

Author Information


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Corresponding Author *E-mail: [email protected]; [email protected] Author Contributions † Z. Feng and P. Tao contributed equally to this work. Funding The authors acknowledge the financial support from National Natural Scientific Foundation of China (51473078, 21671108 and 61605138), National Program for Support of Top-Notch Young Professionals, Scientific and Technological Innovation Teams of Colleges and Universities in Jiangsu Province (TJ215006), Priority Academic Program Development of Jiangsu Higher Education Institutions (YX03001), the Hong Kong Research Grants Council (HKBU12304715) and the Hong Kong Polytechnic University (1-ZE1C), Shanxi Provincial Key Innovative Research Team in Science and Technology (2015013002-10), and the Open Research Fund of State Key Laboratory of Bioelectronics, Southeast University.

Notes The authors declare no competing financial interest.


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Figure 1. Chemical structures, design strategy and synthesis of the phosphorescent polymer dots (Ir-HPC/PSMA dots) for ratiometric hypoxia imaging and the mechanisms of Ir-HPC/PSMA dots in photodynamic therapy.


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Scheme 1. Synthetic route of hyper-branched phosphorescent conjugated polymer Ir-HPC.


20

Normalized Number (a.u.)

b 100

d 40

Absorbance (a.u.)

e

30 20 10 0

0

5

10 15 20 Time (day)

25

30

c

22 nm

Zeta Potential (mV)

a

Average Diameter (nm)

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


80 60 40 20 0 10

1.0

20 30 Diameter (nm)

-2.03 mV Ir-HPC dots

-10

-20

-30

-25.86 mV Ir-HPC/PSMA dots

1.0

Ir-HPC/PSMA dots Ir-HPC in THF

f

Ir-HPC/PSMA dots Ir-HPC in THF

0.8 0.6 0.4 0.2 0.0 300

0

40

Intensity (a.u.)

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0.8 0.6 0.4 0.2 0.0

400 500 600 Wavelength (nm)

700

400

500

600

700

Wavelength (nm)

Figure 2. a) The TEM image of Ir-HPC/PSMA dots; b) DLS of Ir-HPC/PSMA dots in aqueous solution; c) comparison of zeta potentials of Ir-HPC dots and Ir-HPC/PSMA dots in aqueous solution; d) average DLS diameters of Ir-HPC/PSMA dots in aqueous solution for different time periods; e) absorption spectra of Ir-HPC in THF and Ir-HPC/PSMA dots in aqueous solution; f) emission spectra of Ir-HPC in THF and Ir-HPC/PSMA dots in aqueous solution (air, λex = 370 nm, 40 μg·mL-1).


21


750 O2

Intensity (a.u.)

600

0% 2% 4% 6% 8% 10% 15% 21% 30% 40% 50% 60% 70% 80% 90% 100%

O2 contents 0％ air N2

450

100 ％

300

150

b Intensity (Counts)

a

10

3

10

2

10

1

τ = 1114 ns τ = 791 ns τ = 453 ns

N2 Air O2

0 450

500

550

600

650

700

750

0

Wavelength (nm)

c

1.6

d

Original data Trendline

1.5

2000

4000

6000

8000

10000

τ (Delay time) (ns)

1.5


1.4

1.4 1.3

1.3

τ0 / τ

0

RI / R I

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

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1.2

τo / τ = 1 + Ksv[O2]

1.1

0

RI / RI = 1 + Ksv[O2]

1.1

1.2

1.0

1.0 0

5

10

15

20

0

5

O2 contents (%)

10

15

20

O2 content (%)

Figure 3. a) Emission spectra of Ir-HPC/PSMA dots in aqueous solution under different oxygen contents (λex = 370 nm, 40 μg·mL-1); b) luminescence decays of Ir-HPC/PSMA dots at 630 nm in aqueous solution saturated with N2, air and O2, respectively; c) RI0/RI as a function of oxygen contents (R2 = 0.982); d) τ0/τ as a function of oxygen contents (R2 = 0.991).


22

a

Bright field

420-490 nm

600-700 nm

b

Overlay

21% O2

2.5% O2

1.0 0.8 0.6 0.4 0.2 0.0

2.5

21

O2 content (%)

c

PLIM

0-1000 ns

180-1000 ns 270.000

21% O2

2.5% O2

450

Events (Cnts)

Average lifetime (ns)

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


Intensity ratio of red / blue region

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0.000

Figure 4. a) Photoluminescence images; b) plots of ratio of emission intensity; c) photoluminescence lifetime images and time-gated luminescence images with different delay time of HepG2 cells incubated with 8 μg·mL-1 Ir-HPC/PSMA dots under different oxygen contents (21% and 2.5% O2). Collecting ranges were 420-490 nm for polyfluorene emission and 600-700 nm for iridium(III) complex emission, respectively (λex = 405 nm).


23


2.0

b

0 min 3 min 6 min 9 min 12 min 15 min ABDA

1.5

1.0

1.4 0 min 3 min 6 min 9 min 12 min 15 min ABDA

1.2

Absorbance (a.u.)

Absorbance (a.u.)

a

0.5

1.0 0.8 0.6 0.4 0.2 0.0

0.0 350

400

450

350

500

400

Wavelength (nm) Original data Trendline

1.8

450

500

Wavelength (nm)

1.6

1.4

1.2

d Absorbance at 378 nm

c Absorbance at 378 nm

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

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1.2


1.0

0.8

0.6

0.4 0

3

6

9

12

Irradiation time (min)

15

0

3

6

9

12

15

Irradiation time (min)

Figure 5. Absorption spectra of singlet oxygen trapping agent ABDA (200 μM) with (a) Ir-HPC/PSMA dots (40 μg·mL-1) and (b) Ru(ppy)3Cl2 (20 μM) in aqueous solution under irradiation at 420 nm for different irradiation time; plot of the absorbance at 378 nm as a function of light irradiation time in aqueous solution containing (c) Ir-HPC/PSMA dots and ABDA (R2 = 0.989) and (d) Ru(ppy)3Cl2 and ABDA (R2 = 0.996).


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Ir-HPC/PSMA dots DCF 420-490 nm 505-565 nm

Overlay

Bright field

PDT

PDT+VC

After PDT 10 min

Before PDT

a

Laser

Ir-HPC/PSMA dots

PI

FITC

Bright field

b

Overlay

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



25


Figure 6. a) Subcellular localization of ROS generated during Ir-HPC/PSMA dots-mediated PDT by DCFH-DA staining. Cells were incubated with 8 μg·mL-1 Ir-HPC/PSMA dots before irradiation and irradiation of 420 nm light with 10 min (λex = 405 nm). b) Confocal fluorescence images of Annexin V-FITC/PI stained HepG2 cells with different treatments (λex = 488 nm).

c

Bright field

b

Irradiation time

Bright field

a

420-490 nm

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

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Annexin VFITC

Bright field

PI

Annexin V-FITC

Overlay

0 min

30 min

PI

Overlay

Bright field

0h

6h

1h

7h

2h

8h

3h

9h

4h

10 h

5h

11 h

Annexin VFITC

PI

Overlay

Figure 7. a) Photoluminescence images of Ir-HPC/PSMA dots loaded HepG2 cells. λex = 405 nm; b) photoluminescence images of Annexin V-FITC/PI stained Ir-HPC/PSMA dots loaded HepG2 cells with different irradiation times (λex = 488 nm); c) time-lapse photoluminescence images of Annexin V-FITC/PI stained Ir-HPC/PSMA dots loaded HepG2 cells after light irradiation (λex = 488 nm).


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The table of contents The first iridium(III) complex cored phosphorescent conjugated polymer dots with negative charges for ratiometric hypoxia imaging and photodynamic therapy of cancer cells are rational designed and constructed. The dual-emission and phosphorescence nature of nanoprobe enable reliable and sensitive measurements by ratiometric imaging, PLIM and TGLI techniques. The potential applications in image-guided PDT are demonstrated by real-time luminescence imaging.

Keyword:

hyper-branched

polymer

dots,

hypoxia

imaging,

iridium(III)

complexes,

phosphorescence, photodynamic therapy


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The table of contents The first iridium(III) complex cored phosphorescent conjugated polymer dots with negative charges for ratiometric hypoxia imaging and photodynamic therapy of cancer cells are rational designed and constructed. The dual-emission and phosphorescence nature of nanoprobe enable reliable and sensitive measurements by ratiometric imaging, PLIM and TGLI techniques. The potential applications in image-guided PDT are demonstrated by real-time luminescence imaging.


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Hyperbranched Phosphorescent Conjugated Polymer Dots with

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