Cyclometalated Iridium(III)-Complex-Based Micelles for Glutathione

Subscriber access provided by Georgetown University | Lauinger and Blommer Libraries

Article

Cyclometalated Iridium(III) Complex-Based Micelles for GlutathioneResponsive Targeted Chemotherapy and Photodynamic Therapy Xiang Huijing, Hongzhong Chen, Huijun Phoebe Tham, Soo Zeng Fiona Phua, Jin-Gang Liu, and Yanli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09506 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

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

ACS Applied Materials & Interfaces

Cyclometalated Iridium(III) Complex-Based Micelles for GlutathioneResponsive Targeted Chemotherapy and Photodynamic Therapy Huijing Xiang,†,‡,§ Hongzhong Chen,‡,§ Huijun Phoebe Tham,‡ Soo Zeng Fiona Phua,‡ Jin-Gang Liu,*† Yanli Zhao*‡,¶ †

Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East

China University of Science and Technology, Shanghai 200237, P. R. China ‡

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore ¶

School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang

Avenue, Singapore 639798, Singapore Email: [email protected]; [email protected]

KEYWORDS: combination therapy, cyclometalated Ir(III) complex, glutathione activation, micelles, targeted imaging

ABSTRACT. The integration of chemotherapy and photodynamic therapy (PDT) in a single delivery system is highly desirable for enhancing anticancer therapeutic efficacy. Herein, two cyclometalated Ir(III) complex-constructed micelles FIr-1 and FIr-2 were demonstrated for glutathione (GSH) activated targeted chemotherapy and PDT. The cyclometalated Ir(III) complexes

were

prepared

by

conjugating

phosphorescent

Ir(III)

compounds

with

chemotherapeutic drug camptothecin (CPT) through GSH responsive disulfide bond linkages, and the Ir(III) complexes were then assembled with amphiphilic surfactant pluronic F127 via noncovalent encapsulation to afford micelles. The surfaces of the micelles were further decorated

ACS Paragon Plus 1 Environment


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

with folic acid as a targeting group. The micelles showed intense fluorescence that renders them with excellent real-time imaging capability. The release of free anticancer drug CPT from the micelles was realized through GSH-activated disulfide bond cleavage in tumor cells. In addition, the micelles were capable of generating singlet oxygen used for PDT upon visible light irradiation. On account of having folic acid targeting ligand, the micelles displayed greater cellular accumulation in folate receptor (FR) overexpressed HeLa cells than FR low-expressed MCF-7 cells, leading to selective cancer cell killing effect. As compared with solo therapeutic systems, the micelles with targeted combinational chemotherapy and PDT presented superior potency and efficacy in killing tumor cells at a low dosage. On the basis of these findings, the multifunctional micelles could serve as a versatile theranostic nanoplatform for cancer cell targeted imaging and combinational therapy.

1. INTRODUCTION Chemotherapy is a dominant treatment modality for different types and stages of cancer. However, the major challenge in current chemotherapy is how to carry out selective eradication of tumor cells without affecting normal cells and tissues.1,2 Therefore, developing stimulusresponsive drug delivery systems has recently received broad attention as a promising approach to enhance the drug accumulation at targeted tumor sites, thus significantly improving therapeutic outcome of anticancer drugs.3-7 The fact that the concentration of glutathione (GSH) available in tumor cells is 100-1000 folds higher than that in the extracellular microenvironment provides an appropriate opportunity to achieve selective intracellular drug release in tumor cells triggered by this reducing microenvironment.8-12 Thus, improving the therapeutic efficacy and


Page 2 of 28

Page 3 of 28

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


selectivity of anticancer drugs through a smart design of GSH-activatable prodrugs with disulfide bond linkages is highly desirable. Despite remarkable advances in stimulus-responsive drug delivery systems, cancer therapy relying on individual chemotherapy remains suboptimal owing to high dosage requirement, the development of drug resistance, and side effects.13-15 Notably, the combination of multiple therapeutic forms such as integrating chemotherapy with photodynamic therapy (PDT) through different mechanisms of actions can potentially achieve enhanced therapeutic efficacy as compared with a single mode of treatment.16-22 PDT involving the administration of photosensitizers to generate cytotoxic reactive oxygen species (ROS) under light illumination to induce cell apoptosis and necrosis has emerged as an attractive clinical modality for cancer treatment owing to its noninvasive feature and improved selectivity.23-25 Hematoporphyrin derivatives are the most commonly used photosensitizers for PDT. However, their poor ROS generation under hypoxia conditions, low molar absorption coefficient, and side effects have limited the PDT efficacy for clinical uses.26-28 Cyclometalated Ir(III) complexes as efficient photosensitizers have been developed to overcome these limitations.29-34 In addition, unique properties of phosphorescent cyclometalated Ir(III) complexes, including large Stokes’ shifts, high quantum yields, good resistance to photobleaching, and cell permeability, make them ideal candidates as bioimaging agents.35-42 Recently, several studies have reported that cyclometalated Ir(III) complexes can function as efficient photosensitizers for both PDT and bioimaging.29-34 To the best of our knowledge, however, there was no study on the development of GSH activated cytometalated Ir(III) complexes bearing anticancer drugs through disulfide bond linkages for combinational chemotherapy and PDT accompanied by bioimaging. Therefore, integrating the cyclometalated



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

Ir(III) complexes with functions of anticancer drug release and ROS generation in a single delivery system offers a great opportunity for the construction of novel theranostic platforms.

Scheme 1. (A) Chemical structures of the Ir(III) compounds covalently linked with anticancer drug CPT through GSH-cleavable linkers, and schematic illustration for the formations of FIr-1 and FIr-2 via nanoprecipitation using pluronic F127-FA and Ir(III) compounds Ir-1 and Ir-2. (B) Schematic illustration of the ROS generation upon visible light illumination for PDT and anticancer drug release triggered by GSH for chemotherapy in tumor cells.


Page 4 of 28

Page 5 of 28

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


Herein, we report a facile strategy to fabricate two GSH-activatable Ir(III) complexes (Ir(ppy)2(bpy-CPT) (Ir-1) and Ir(Qpy)2(bpy-CPT) (Ir-2)) and corresponding amphiphilic surfactant (Pluronic F127-FA) encapsulated micelles (FIr-1 and FIr-2) for targeted imaging, chemotherapy and PDT (Scheme 1). By covalently conjugating chemotherapeutic drug camptothecin (CPT) with Ir(III) complexes through disulfide linkers, GSH-responsive photosensitizers Ir-1 and Ir-2 were obtained. Meanwhile, visible light irradiation could activate the Ir(III) complexes to generate ROS for PDT. Due to poor water solubility of Ir(III) complexes in biological conditions, the encapsulation of Ir(III) complexes by Pluronic F127-FA afforded applicable micelles FIr-1 and FIr-2. Furthermore, the micelles functionalized with the folic acid (FA) targeting ligand could lead to enhanced cellular uptake via folate receptor mediated endocytosis.43 The triple combination of targeted delivery, GSH-triggered anticancer drug CPT release in tumor microenvironment, and PDT under visible light irradiation is highly beneficial to the optimization of therapeutic efficacy in the tumor treatment.

Scheme 2. Synthetic route of the cyclometalated Ir(III) complexes Ir-1, Ir-2, Ir-3 and Ir-4.



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

2. RESULTS AND DISCUSSION Two different ligands, 2-phenylpyridine (ppy) and 2-phenylquinoline (pq), were incorporated into the Ir(III) center. Then, the bipyridine (bpy) ligand and bpy functionalized with chemotherapeutic drug CPT (bpy-CPT) ligand were employed as ancillary ligands to afford final cationic Ir(III) complexes with high emission quantum yields.44 Four cationic Ir(III) complexes Ir(ppy)2(bpy-CPT)+ (Ir-1), Ir(pq)2(bpy-CPT)+ (Ir-2), Ir(ppy)2(bpy)+ (Ir-3), and Ir(pq)2(bpy)+ (Ir4) were synthesized accordingly (Scheme 2). Ir-1 and Ir-2 were developed as efficient photosensitizers and prodrugs, while Ir-3 and Ir-4 were designed as contrast photosensitizers. The synthesis and characterization of the Ir(III) complexes, including Ir-1, Ir-2, Ir-3 and Ir-4, are summarized in the Supporting Information (Scheme S1, Figures S1 and S2, and Table S1). As expected, the Ir(III) complexes exhibited metal-to-ligand charge transfer (MLCT), ranging from 350 nm to 500 nm (Figure S1 and Table S1). Their phosphorescence emission bands locate in the range of 480-650 nm (Table S1). Varied phosphorescence lifetimes and quantum yields were observed for these four Ir(III) complexes, suggesting their tunable emission properties. To achieve good dispersibility, water solubility, and biocompatibility of cyclometalated Ir(III) complexes, an amphiphilic surfactant Pluronic F127-FA containing a targeting moiety FA was employed to encapsulate the Ir(III) complexes to obtain phosphorescent micelles FIr-1, FIr-2, FIr-3, and FIr-4. Transmission electron microscopy (TEM) was used to study the morphology of these micelles (Figure 1A-D), revealing that as-prepared micelles showed a regular spherical shape with average diameters of 140, 75, 136, and 110 nm, respectively. The hydrodynamic diameters of the micelles were investigated by dynamic light scattering (DLS), which displayed average hydrodynamic diameters of 159, 90, 168, and 121 nm, respectively (Figure S3), quite close to those derived from TEM studies. A negative apparent zeta potential value of -10.55 ±


Page 6 of 28

Page 7 of 28

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


0.59 mV for F127-FA (Figure 1E) confirms that the FA modified F127 is negatively charged on account of rich carboxylic groups on F127-FA. Owing to the cationic property of the Ir(III) complexes, the micelles FIr-1, FIr-2, FIr-3 and FIr-4 had similar positive zeta potentials of 1.79 ± 0.21 mV, 1.4 ± 0.61 mV, 2.35 ± 0.14 mV, and 0.97 ± 0.27 mV, respectively.

Figure 1. TEM images of the micelles (A) FIr-1, (B) FIr-2, (C) FIr-3, and (D) FIr-4. Scale bars: 100 nm. (E) Zeta potential of F127-FA and the micelles FIr-1, FIr-2, FIr-3 and FIr-4. (F) FT-IR spectra of F127-FA and the micelles FIr-1, FIr-2, FIr-3 and FIr-4.

FT-IR spectra of F127-FA and the micelles are illustrated in Figure 1F. The FT-IR spectrum of F127-FA exhibited the characteristic bands at 3420, 2900, 1640 and 1100 cm−1, which can be assigned to O-H or N-H, C-H, C=O and C-O stretching respectively, suggesting successful anchoring of FA to the surfactant F127. Meanwhile, the micelles FIr-1, FIr-2, FIr-3 and FIr-4 displayed dominant characteristic bands of F127-FA, indicating the encapsulation of



iridium complexes inside the micelles using amphiphilic surfactant F127-FA. The micelles were further characterized by energy dispersive spectrometer (EDS). The EDS spectra (Figures S4 and S5) clearly revealed that only elements Ir, C and O were detected in the micelles FIr-1, FIr-2, FIr-3 and FIr-4.

A

F Ir-4 F Ir-2

B

C

0.9

0.6 0.3

0.6

0.9

Ir-3 Ir-1 Ir-4 Ir-2

0.3

0.0 4 00 500 W a v ele ngth (nm )

60 0

60 0 F Ir-3 F Ir-1

45 0

0.0 30 0

4 00 500 W a v ele ngth (nm )

60 0

F Ir-4 F Ir-2

45 0

15 0 0 500

600 700 W a v ele ngth (nm )

80 0

60 0

E

F

30 0

30 0

D

F luore s c e nc e Intens ity (a .u.)

30 0

A bs orba nc e (a .u.)

1.2 A bs orba nc e (a .u.)

F Ir-3 F Ir-1

1.2

F luore s c e nc e Intens ity (a .u.)

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 8 of 28

Ir-3 Ir-1 Ir-4 Ir-2

15 0 0 500

600 7 00 W av e leng h (nm)

80 0

Figure 2. UV-Vis absorption spectra of (A) FIr-1 and FIr-3 and (B) FIr-2 and FIr-4 in PBS solution (pH = 7.4, 10 mM). Fluorescence spectra of (D) FIr-1 and FIr-3 and (E) FIr-2 and FIr-4 in PBS solution (pH = 7.4, 10 mM). Photographs of corresponding samples in PBS solution (pH = 7.4, 10mM) (C) before and (F) after excited by a UV lamp (the wavelength is 365 nm).

The UV-Vis absorption of the micelles encapsulated with cytometalated Ir(III) complexes in phosphate buffered saline (PBS) solution was also determined. As illustrated in Figure 2A,B, the micelles exhibited absorption maxima at 361, 377 and 411 nm for FIr-1, 323, 348 and 433 nm for FIr-2, 383 and 417 nm for FIr-3, and 339 and 440 nm for FIr-4. The micelles FIr-1, FIr-2, FIr-3 and FIr-4 displayed intense absorption bands in the range of 300-400 nm, which could be


Page 9 of 28

assigned to spin-allowed ligand-to-ligand charge transfer (LLCT) transitions. Weak absorption bands appeared in the visible light range of 400-450 nm were attributed to mixed singlet and triplet metal-to-ligand charge transfer (1MLCT and 3MLCT) transitions.45 The maximum emission wavelengths of the micelles were conducted by fluorescence spectroscopy, which are at 480 and 640 nm for FIr-1, 610 nm for FIr-2, 566 nm for FIr-3, and 553 nm for FIr-4 when excited at the MLCT (Figure 2D,E). The emission spectrum of FIr-1 located at 480 nm was dominated by the ligand bpy-CPT. The photographs of the micelles FIr-1, FIr-2, FIr-3 and FIr-4 in vials before and after UV lamp irradiation are shown in Figure 2C,F, exhibiting blue, red, yellow, and green fluorescence, respectively.

5 m in

0.4

350

4 00 450 5 00 W a v ele ngth (nm )

55 0

30 0


0.9

0

0.6 0.3

5 m in

0.0

350

4 00 450 5 00 W a ve le ngth (nm )

4 00 450 5 00 W a ve le ngth (nm )

55 0

E

0.9

0

5 min

30 0

350

4 00 450 5 00 W a v ele ngth (nm )

55 0

F

0.9

0.6

0.6

0.3

5 m in

0.3

30 0

0.3

55 0

0.0 350

0.6

0.0

1.2

D

1.2

30 0

5 m in

0.3 0.0

0.0 30 0

0.6

0

0.8

0

C

0.9

0

B

0.9

1.2

C /C 0 (@ 4 1 7 nm)



A

1.2

1.2


1.6


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


F Ir-1

F Ir-2 F Ir-3 F Ir-4

0.0 350

4 00 450 5 00 W a v ele ngth (nm )

55 0

0

1

D PB F

M B

2 3 T im e (min)

4

5

Figure 3. Absorbance of DPBF after photodecomposition by 1O2 generated under visible light irradiation (> 400 nm, 100 mW cm-2) over different periods of time in the case of (A) control, (B) FIr-1, (C) FIr-2, (D) FIr-3, and (E) FIr-4 in PBS solution (10 mM, pH 7.4). (F) Normalized absorbance changes of DPBF at 417 nm during photodecomposition by 1O2 generation for



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

control (DPBF), micelles FIr-1, FIr-2, FIr-3, FIr-4, and MB in PBS solution (10 mM, pH 7.4) under visible light irradiation (> 400 nm, 100 mW cm-2).

The singlet oxygen (1O2) generation efficiency of the micelles FIr-1, FIr-2, FIr-3 and FIr-4 under visible light irradiation was performed by monitoring the time-dependent absorbance of 1,3-diphenylisobenzofuran (DPBF) at 417 nm. As we known, DPBF is an efficient 1O2 scavenger that possesses a highly specific reactivity toward 1O2 forming its oxidized product 1,2dibenzoylbenzene.46,47 The absorption spectra of DPBF in the presence of the micelles FIr-1, FIr2, FIr-3, and FIr-4 with increasing exposure time are illustrated in Figure 3B-E, and corresponding absorbance variations are shown in Figure 3F. Figure 3A presents the absorption intensity of DPBF without any photosensitizer as a function of irradiation time. A negligible change of the DPBF absorbance in PBS solution was observed within 5 min, suggesting that DPBF was stable under the experimental conditions. After the addition of the micelle FIr-1, the characteristic absorption peak of DPBF at 417 nm gradually decreased within 5 min under visible light irradiation (Figure 3B). Similar observations were achieved when using the micelles FIr-2 and FIr-3 as photosensitizers under the same experimental conditions. Excitingly, the absorption peak of DPBF significantly decreased within 5 min when the micelle FIr-4 was employed (Figure 3E). Therefore, the micelle FIr-4 possesses the highest efficiency of 1O2 generation among the four micelles. Furthermore, the quantum yields for 1O2 production (ΦΔ) from these micelles, as a key parameter for the evaluation of the 1O2 generation efficiency by a photosensitizer upon photoexcitation, were obtained by measuring the time-dependent photodegradation of DPBF (Figure 3F). The ΦΔ values of FIr-1, FIr-2, FIr-3 and FIr-4 were


Page 10 of 28

Page 11 of 28

determined to be 0.133, 0.194, 0.164 and 0.274 respectively, using methylene blue (MB) as the standard (ΦΔ = 0.55).

A

0 m M

0.1 mM

1 m M

80 60

40 20 0 0

10

20 30 T ime (h)

40

50

100

B

0 mM

0.1 mM

1 mM

80 60

100

C umulative C P T rele as e (% )

C um ula tive C P T re lea s e (% )

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


40 20 0 0

10

20 30 T ime (h)

40

50

Figure 4. CPT release profiles from the micelles (A) FIr-1 (0.1 mM) and (B) FIr-2 (0.1 mM) as a function of time in the absence and presence of GSH (0.1 and 1 mM). All data were measured at 37 oC in PBS solution (10 mM, pH 7.4).

We next investigated the chemotherapeutic drug CPT release bebavior of the micelles FIr-1 and FIr-2 in response to reducing agents. First, we compared the CPT release efficiency of FIr-1 in the presence of different concentrations of GSH. As seen from Figure 4A, after 48 h of incubation, 53.7% and 83.1% of CPT were released in the presence of 0.1 and 1 mM GSH respectively, while negligible CPT release was observed in the absence of GSH, demonstrating GSH-responsive CPT release property of FIr-1. FIr-2 displayed a similar CPT release profile to FIr-1, releasing 55.8% and 86.5% of CPT in the presence of 0.1 and 1 mM GSH within 48 h, respectively (Figure 4B). To further confirm the cleavage of activatable disulfide linker upon the GSH treatment, UV spectral changes of the micelles FIr-1 and FIr-2 treated with 10 equiv. GSH were monitored. As shown in Figure S6, it was observed that absorption bands at 362 and 378 nm shifted to 390 nm for FIr-1, and 433 nm to 390 nm for FIr-2. It was also found that the



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

emission spectra of FIr-1 and FIr-2 underwent blue shifts to 410 and 435nm for FIr-1, as well as 436 and 460 nm for FIr-2. There were no substantial changes in the phosphorescence lifetime for FIr-1 and FIr-2 after the treatment with GSH.

Figure 5. Confocal microscopy images of HeLa and MCF-7 cells treated with (A) FIr-1 (5 µM), (B) FIr-2 (5 µM), (C) FIr-3 (5 µM), and (D) FIr-4 (5 µM) for 2 h. The samples were excited at 405 nm and detected at 460-510 nm for FIr-1, 580-630 nm for FIr-2, 550-580 nm for FIr-3, and 520-570 nm for FIr-4. Scale bar = 30 µm. Flow cytometry analysis of HeLa cells treated with (E,


Page 12 of 28

Page 13 of 28

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


I) FIr-1 (5 µM), (F, J) FIr-2 (5 µM), (G, K) FIr-3 (5 µM), and (H, L) FIr-4 (5 µM) for 2 h. HeLa cells treated with the micelles: blue lines, MCF-7 cells treated with the micelles: green lines, untreated HeLa cells: black lines, and untreated MCF-7 cells: red lines. M means MCF-7 cells, and H means HeLa cells. Single asterisk symbol (*) represents p < 0.05, and double asterisk symbol (**) represents p < 0.01.

Images of HeLa cells upon the incubation with each micelle were obtained by confocal microscopy. All the micelles emitted noticeable fluorescence even at a relatively low concentration (5 µM) upon the incubation for 2 h. To demonstrate the role of the FA moiety in guiding the micelles to folate receptor-rich tumor cells, the micelles FIr-1, FIr-2, FIr-3 and FIr-4 were incubated with two different cancer cell lines, namely HeLa and MCF-7 cells. The two cell lines were chosen because the expression level of the folate receptor in HeLa cells is much higher than that in MCF-7 cells. As seen from Figure 5A-D, HeLa cells treated with micelles displayed strong fluorescence upon the excitation at 405 nm. In the case of MCF-7 cells, under similar experimental conditions, relatively weak fluorescence was observed for each micelle. Cellular internalization was then investigated using flow cytometry to confirm and quantify the enhanced cellular uptake of these micelles. As presented in Figure 5E-L, HeLa cells treated with the micelles yielded intense fluorescence as compared with MCF-7 cells, approximately 2.1-fold for FIr-1, 3.4-fold for FIr-2, 3.2-fold for FIr-3, and 1.8-fold for Ir-4. In addition, inductively coupled plasma mass spectrometry (ICP-MS) analysis was carried out to quantitatively determine the cellular uptake of the micelles FIr-1, FIr-2, FIr-3 and FIr-4. Under the same experimental conditions, the uptake amounts of iridium content in HeLa vs MCF-7 cells were 5.8 ± 0.4 vs 2.5 ± 0.2 ng/cell for FIr-1, 7.1 ± 0.60 vs 2.8 ± 0.2 ng/cell for FIr-2, 6.4 ± 0.8 vs 3.3 ± 0.4



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

ng/cell for FIr-3, and 5.6 ± 0.3 vs 2.6 ± 0.5 ng/cell for FIr-4. Enhanced cellular uptake observed with HeLa cells incubated with these micelles could be ascribed to the presence of folate receptor mediated endocytosis of the micelles in cancer cells, guided by the interactions of FA moiety with folate receptor on the surface of tumor cells. Thus, flow cytometry studies are consistent with confocal microscopy results that the micelles modified with FA led to enhanced cellular uptake and selective accumulation in folate receptor overexpressed cancer cells via receptor-mediated endocytosis.

Figure 6. DCF fluorescence in HeLa cells treated with (A,E) FIr-1, (B,F) FIr-2, (C,G) FIr-3, and (D,H) FIr-4. HeLa cells without any treatment: black line, HeLa cells treated with visible light for 5 min: green line, HeLa cells treated with micelles (5 µM) in the dark: blue line, and HeLa cells treated with micelles (5 µM) under visible light irradiation (> 400 nm, 100 mW cm-2) for 5 min: red line. Single asterisk symbol (*) represents p < 0.05, and double asterisk symbol (**) represents p < 0.01.


Page 14 of 28

Page 15 of 28

Intracellular ROS generation of the micelles FIr-1, FIr-2, FIr-3 and FIr-4 under visible light irradiation was validated by using a cell permeable ROS fluorescent probe 2′,7′dichlorofluorescin diacetate (DCF-DA). DCF-DA is nonfluorescent and easily oxidized by ROS to fluorescent dichlorofluorescein.48 As shown in Figure 6, the incubation of the micelles without visible light irradiation gave very weak fluorescence of DCF. Meanwhile, negligible ROS generation was detected when HeLa cells were treated with visible light irradiation alone. When HeLa cells were incubated with each micelle for 4 h and then treated with visible light irradiation for 5 min, increased fluorescence was observed as compared with the cases in the dark, approximately 258-fold increase for FIr-1, 191-fold for FIr-2, 120-fold for FIr-3, and 30-fold for FIr-4, indicating efficient production of intracellular ROS under the irradiation with visible light. F Ir-3

F Ir-1

*

F Ir-4

F Ir-2

*

*

12 0

40 20

0

**

40

0 80

** **

*

**

**

0 0.5 1 2 4 C onc entration ( µM) T a rg e te d m ic e lle s N on -ta rg e t m ic e lle s

60

*

* *

40 20

**

t l t T t 1 t 4 t ro lig h C P I r- 3 lig h I r- ig h I r- lig h I r- 2 lig h t l F 4+ F + F + F + o n l+ 2 1 -3 C tro rI rr F Ir I n F FI F o C

100 2.73796 100 2.33258 78.93552 3.50975 52.9079 69.56769 5.77391 40.9616 57.75 6.7631 29.17048 0.97352 50.79 0.73988 13.07402

*

C ell v iability (% )

C

60

20

*

60

80

40

*

80

0 0 .5 1 2 4 C onc entration ( µM)

10 0

*

F Ir-1 _ lig ht F Ir-2 _ lig ht

20

0 12 0

10 0

80 60

F Ir-3 _lig ht F Ir-4 _lig ht

B

A


10 0


12 0


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


0

F Ir-1 F Ir-2 F Ir-3 F Ir-4



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

Figure 7. Viability of HeLa cells treated with micelles FIr-1, FIr-2, FIr-3 and FIr-4 (0-4 µM) (A) in the dark and (B) under visible light irradiation (> 400 nm, 100 mW cm-2, 10 min). (C) Viability of HeLa cells without any treatment (control), under visible light irradiation (control + light), treated with CPT and the micelle FIr-1, FIr-2, FIr-3 and FIr-4, and treated with the micelle FIr-1, FIr-2, FIr-3 and FIr-4 under visible light irradiation. (D) Viability of HeLa cells treated with the micelles FIr-1, FIr-2, FIr-3, and FIr-4, and the non-targeted micelles NFIr-1, NFIr-2, NFIr-3, and NFIr-4 under visible light irradiation (> 400 nm, 100 mW cm-2, 10 min). Single asterisk symbol (*) represents p < 0.05, and double asterisk symbol (**) represents p < 0.01.

To evaluate the feasibility of the micelles FIr-1 and FIr-2 for combined photodynamic and chemotherapy, the cytotoxicity of the micelles FIr-1, FIr-2, FIr-3 and FIr-4 was analyzed with the 3-(4,5-dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and MCF-7 cell lines in the dark or under visible light irradiation. The treatment of HeLa cells only with visible light (> 400 nm, 100 mW cm-2, 10 min) showed negligible toxicity to HeLa cells. The cell viability of HeLa cells treated with different concentrations of CPT is shown in Figure S7. As present in Figure 7A, in the absence of visible light irradiation, the micelles FIr-3 and FIr4 did not exhibit detectable cytotoxicity for HeLa cells. In contrast, the micelles FIr-3 and FIr-4 under visible light irradiation showed obvious cytotoxicity to HeLa cells, confirming their PDT effect (Figure 7B). Higher cell viability was observed when HEK 293 cells were incubated with the micelles FIr-3 and FIr-4 under same experimental conditions (Figure S8). When HeLa cells were incubated with the micelles FIr-1 and FIr-2 in the dark, the cell viability obviously decreased as increasing the micelle concentrations. This was due to the released CPT triggered by high GSH concentration in tumor cells for chemotherapy of cancer. The highest cell death


Page 16 of 28

Page 17 of 28

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


rate was achieved with the combined action of CPT release triggered by high GSH concentration and singlet oxygen generation upon visible light irradiation, when HeLa cells were incubated with the micelles FIr-1 or FIr-2 under visible light irradiation. For example, the cell viability of FIr-2 under visible light irradiation is 13.07% at the concentration of 4 µM, showing much higher cytotoxicity than that of the FIr-2 group (36.09 %) incubated with HeLa cells in the dark and that of the irradiated FIr-4 group (50.79%, control group for FIr-2) at the same concentration. A similar observation was achieved for FIr-1 under these experimental conditions (Figure 7C). The results indicate that the combined chemotherapy and PDT by the micelle FIr-1 or FIr-2 could effectively destroy cancer cells upon visible light irradiation. Notably, we observed obviously higher cell viability of the non-targeted micelle NFIr-1, NFIr-2, NFIr-3 or NFIr-4 incubated with HeLa cells upon visible light irradiation as compared with that of the FA targeted micelle FIr-1, FIr-2, FIr-3 or FIr-4 under similar experimental conditions (Figures 7D and S9). This observation could be rationalized to a significant increase in the number of micelles accumulated in folate receptor overexpressed HeLa cells via receptor mediated endocytosis, thereby leading to higher cytotoxicity of HeLa cells. Collectively, these results demonstrate that the targeted micelles FIr-1 and FIr-2 could serve as robust therapeutic agents to achieve significant anticancer efficacy by synergistic photodynamic and chemotherapy.

3. CONCLUSIONS In summary, GSH-responsive cyclometalated Ir(III) complexes encapsulated by amphiphilic micelles and further modified with targeting moiety FA have been demonstrated as versatile nanoplatforms (FIr-1, FIr-2, FIr-3 and FIr-4) for synergistic chemotherapy and PDT of cancer cells. GSH-activated Ir(III) complexes have been constructed by integrating Ir(III) compounds



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

with anticancer drug CPT via disulfide bond linker. The disulfide bond can be cleaved by high GSH concentration in tumor cells to release free anticancer drug CPT for chemotherapy. In addition, these micelles could efficiently generate singlet oxygen upon visible light irradiation. It has also been demonstrated that outstanding imaging capability was achieved through inherent fluorescence of these micelles in vitro. Furthermore, these micelles containing FA targeting ligands have exhibited higher cellular uptake in HeLa cells as compared with normal cells, resulting in selective cancer cell killing effect. More importantly, significantly enhanced therapeutic efficacy has been achieved by the micelles FIr-1 and FIr-2 via an effective combination of chemotherapy and PDT under visible light irradiation. Based on these exciting results, we anticipate that multifunctional micelles FIr-1 and FIr-2 would offer a versatile nanoplatform for simultaneous bioimaging and highly efficient combinational tumor therapy.

4. EXPRIMENTAL METHODS Preparation of the micelles FIr-1, FIr-2, FIr-3, and FIr-4. Briefly, cytometalated Ir(III) complexes (1 mg mL-1) and concentrated F127-FA surfactant (0.1 mM) were dissolved in dicholormethane (1 mL) in a round-bottom flask. PBS (10 mL) at pH 7.4 was then added to the mixture, and the obtained solution was sonicated for 30 min. Complete evaporation of dichloromethane led to the formation of Ir(III) complex-encapsulated micelles. As-prepared micelles were dialyzed against deionized water for 2 days in order to remove any impurities and big particles. Singlet oxygen generation of the micelles FIr-1, FIr-2, FIr-3, and FIr-4. Singlet oxygen (1O2) generation of the micelles FIr-1, FIr-2, FIr-3, and FIr-4 was determined by using DPBF as a chemical 1O2 probe. In a typical experiment, FIr-1, FIr-2, FIr-3, or FIr-4 in PBS containing


Page 18 of 28

Page 19 of 28

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


DPBF was irradiated with visible light laser at the power density of 100 mW cm-2 for different periods of time, and the absorbance of DPBF at 417 nm was measured. PBS containing DPBF was also prepared as a control. Confocal microscopy studies of the micelle uptake. The uptake of the micelles by HeLa cells and MCF-7 cells was investigated using confocal microscopy. HeLa cells (5 × 104 cells/well) and MCF-7 cells (5 × 104 cells/well) were seeded in a 6-well plate and incubated at 37 oC in a humidified atmosphere for 24 h. The micelles were then added to the cells, which were incubated for another 2 h. Subsequently, the cells were washed three times with PBS. Finally, the cells were imaged by ZEISS LSM 800 confocal laser scanning microscope with laser excitation at 405 nm. Flow cytometry studies of the micelle uptake. For receptor-mediated endocytosis experiments, HeLa and MCF-7 cells were incubated with the micelle solution (5 µM) in cell culture medium at 37 oC for 2 h. Then, the cells were washed three times with PBS and harvested by trypsinization, followed by centrifugation at 2000 rpm for 5 min. The precipitate thus obtained was re-suspended in PBS and analyzed using a flow cytometer. Flow cytometry studies of intracellular 1O2 detection. HeLa cells were incubated with the micelles (FIr-1, FIr-2, FIr-3, and FIr-4) in cell culture medium at 37 oC for 6 h, respectively. The cells were washed twice with PBS, treated with DCFH-DA (10 µM) and then incubated for another 20 min. After visible light irradiation (λ > 400 nm, 100 mW/cm2, 5 min), the cells were washed three times with PBS and detached by trypinization, followed by centrifugation at 2000 rpm for 5 min. The cells thus obtained were re-suspended in PBS and analyzed using a flow cytometer.



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

MTT assays. The cytotoxicity of the micelles (FIr-1, FIr-2, FIr-3, and FIr-4) was evaluated on HeLa cells and MCF-7 cells using MTT assays, respectively. The cells (105 cells/well) were seeded in a 96-well plate and incubated for 24 h. The medium was removed and then the samples were added with fresh culture medium containing the micelles FIr-1, FIr-2, FIr-3, or FIr-4 at final concentrations of 0-4 µM. The medium was removed after incubating for 24 h, and then the cells were washed with PBS twice. Subsequently, fresh medium and MTT solution were added and the obtained samples were incubated for further 4 h. The absorbance at 570 nm for each well was determined by a microplate reader. After incubating the cells with different concentrations of the micelles (0, 0.5, 1, 2, and 4 µM) for 4 h respectively, light irradiation was applied (λ > 400nm, 100 mW/cm2, 10 min), and the cells were further incubated for 24 h. Subsequently, the same procedures, as described above, were performed to conduct the absorbance measurements at 570 nm using a microplate reader.

Supporting Information Detailed synthesis of the ligand bpy-CPT and Ir(III) complexes Ir-1, Ir-2, Ir-3 and Ir-4, photophysical studies, cyclic voltammetry, DLS, EDS, MTT assays, and 1H NMR spectra.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] Notes §

These authors contributed equally to this work. The authors declare no competing financial

interest.


Page 20 of 28

Page 21 of 28

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


ACKNOWLEDGMENT We are grateful for the financial support from the National Nature Science Foundation of China (21571062 and 21628401), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, the Fundamental Research Funds for the Central Universities (No. 222201717003), and the SingHealth-NTU Research Collaborative Grant (No. SHS-NTU/009/2016). H. J. Xiang acknowledges the scholarship support from the Chinese Scholarship Council (No. 201506740024).

REFERENCES 1. Atkins, J. H.; Gershell, L. J. Selective Anticancer Drugs. Nat. Rev. Cancer 2002, 2 (6), 645–646. 2. Umar, A.; Dunn, B. K.; Greenwald, P. Future Directions in Cancer Prevention. Nat. Rev. Cancer 2012, 12 (12), 835–848. 3. Noh, J.; Kwon, B.; Han, E.; Park, M.; Yang, W.; Cho, W.; Yoo, W.; Khang, G.; Lee, D. Amplification of Oxidative Stress by a Dual Stimuli-Responsive Hybrid Drug Enhances Cancer Cell Death. Nat. Commun. 2015, 6, 6907–6916. 4. Fan, F.; Wang, L.; Li, F.; Fu, Y.; Xu, H. Stimuli-Responsive Layer-by-Layer TelluriumContaining Polymer Films for the Combination of Chemotherapy and PDT. ACS Appl. Mater. Interfaces 2016, 8 (26), 17004–17010. 5.

Ma, X.; Zhao, Y. Biomedical Applications of Supramolecular Systems Based on Host–

Guest Interactions. Chem. Rev. 2015, 115 (15), 7794–7839. 6. Nguyen, K. T.; Zhao, Y. Engineered Hybrid Nanoparticles for On-Demand Diagnostics and Therapeutics. Acc. Chem. Res. 2015, 48 (12), 3016–3025.



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

7. Karimi, M.; Ghasemi, A.; Zangabad, P. S.; Rahighi, R.; Basri, S. M. M.; Mirshekari, H.; Amiri, M.; Pishabad, Z. S.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.; Aref, A. R.; Haghani, L.; Bahrami, S.; Hamblin, M. R. Smart Micro/Nanoparticles in StimulusResponsive Drug/Gene Delivery Systems. Chem. Soc. Rev. 2016, 45 (5), 1457–1501. 8. Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T. D.; Tian, H.; Zhu, W. In Vivo and in Situ Tracking Cancer Chemotherapy by Highly Photostable NIR Fluorescent Theranostic Prodrug. J. Am. Chem. Soc. 2014, 136 (9), 3579–3588. 9.

Kong, F.; Liang, Z.; Luan, D.; Liu, X.; Xu, K.; Tang, B. A Glutathione (GSH)-Responsive

Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging. Anal. Chem. 2016, 88 (12), 6450–6456. 10. Wang, Y.; Zhu, L.; Wang, Y.; Li, L.; Lu, Y.; Shen, L.; Zhang, L. W. Ultrasensitive GSHResponsive Ditelluride-Containing Poly(Ether-Urethane) Nanoparticles for Controlled Drug Release. ACS Appl. Mater. Interfaces 2016, 8 (51), 35106–35113. 11. Xu, X.-D.; Zhao, L.; Qu, Q.; Wang, J.-G.; Shi, H.; Zhao, Y. Imaging-Guided Drug Release from Glutathione-Responsive Supramolecular Porphysome Nanovesicles. ACS Appl. Mater. Interfaces 2015, 7 (31), 17371–17380. 12. Chen, H.; Tham, H. P.; Ang, C. Y.; Qu, Q.; Zhao, L.; Xing, P.; Bai, L.; Tan, S. Y.; Zhao, Y. Responsive Prodrug Self-Assembled Vesicles for Targeted Chemotherapy in Combination with Intracellular Imaging. ACS Appl. Mater. Interfaces 2016, 8 (37), 24319–24324. 13. Zhang, C.-J.; Hu, Q.; Feng, G.; Zhang, R.; Yuan, Y.; Lu, X.; Liu, B. Image-Guided Combination Chemotherapy and Photodynamic Therapy Using a Mitochondria Targeted Molecular Probe with Aggregation Induced Emission Characteristics. Chem. Sci. 2015, 6 (8), 4580–4586.


Page 22 of 28

Page 23 of 28

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


14. Feng, L.; Cheng, L.; Dong, Z.; Tao, D.; Barnhart, T. E.; Cai, W.; Chen, M.; Liu, Z. Theranostic Liposomes with Hypoxia-Activated Prodrug to Effectively Destruct Hypoxic Tumors Post-Photodynamic Therapy. ACS Nano 2017, 11 (1), 927–937. 15. He, C.; Liu, D.; Lin, W. Self-Assembled Core-Shell Nanoparticles for Combined Chemotherapy and Photodynamic Therapy of Resistant Head and Neck Cancers. ACS Nano 2015, 9 (1), 991–1003. 16. Xiang, H.-J.; An, L.; Tang, W.-W.; Yang, S.-P.; Liu, J.-G. Photo-Controlled Targeted Intracellular Delivery of Both Nitric Oxide and Singlet Oxygen Using a Fluorescence-Trackable Ruthenium Nitrosyl Functional Nanoplatform. Chem. Commun. 2015, 51 (13), 2555–2558. 17. Xiang, H.-J.; Deng, Q.; An, L.; Guo, M.; Yang, S.-P.; Liu, J.-G. Tumor Cell Specific and Lysosome-Targeted Delivery of Nitric Oxide for Enhanced Photodynamic Therapy Triggered by 808 nm Near-Infrared Light. Chem. Commun. 2016, 52 (1), 148–151. 18. Xiang, H.-J.; Guo, M.; An, L.; Yang, S.-P.; Q.-L. Zhang, Liu, J.-G. A Multifunctional Nanoplatform for Lysosome Targeted Delivery of Nitric Oxide and Photothermal Therapy Under 808 nm Near-Infrared Light. J. Mater. Chem. B 2016, 4 (27), 4667–4674. 19. Zhang, Y.; Ang, C. Y.; Li, M.; Tan, S. Y.; Qu, Q.; Zhao, Y. Polymeric Prodrug Grafted Hollow Mesoporous Silica Nanoparticles Encapsulating Near-Infrared Absorbing Dye for Potent Combined Photothermal-Chemotherapy. ACS Appl. Mater. Interfaces 2016, 8 (11), 6869–6879. 20. Zhang, Y.; Teh, C.; Li, M.; Ang, C. Y.; Tan, S. Y.; Qu, Q.; Korzh, V.; Zhao, Y. AcidResponsive Polymeric Doxorubicin Prodrug Nanoparticles Encapsulating a Near-Infrared Dye for Combined Photothermal-Chemotherapy. Chem. Mater. 2016, 28 (19), 7039–7050.



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

21. Tham, H. P.; Chen, H.; Tan, Y. H.; Qu, Q.; Sreejith, S.; Zhao, L.; Venkatraman, S. S.; Zhao, Y. Photosensitizer Anchored Gold Nanorods for Targeted Combinational Photothermal and Photodynamic Therapy. Chem. Commun. 2016, 52 (57), 8854–8857. 22. Li, M.; Teh, C.; Ang, C. Y.; Tan, S. Y.; Luo, Z.; Qu, Q.; Zhang, Y.; Korzh, V.; Zhao, Y. Near-Infrared Light-Absorptive Stealth Liposomes for Localized Photothermal Ablation of Tumors Combined with Chemotherapy. Adv. Funct. Mater. 2015, 25 (35), 5602–5610. 23. Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T. Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization. Chem. Rev. 2010, 110 (5), 2795–2838. 24. Lovell, J. F.; Liu, T. W. B.; Chen, J.; Zheng, G. Activatable Photosensitizers for Imaging and Therapy. Chem. Rev. 2010, 110 (5), 2839-2857. 25. Zhou, Z.; Song, J.; Nie, L.; Chen, X. Reactive Oxygen Species Generating Systems Meeting Challenges of Photodynamic Cancer Therapy. Chem. Soc. Rev. 2016, 45 (23), 6597– 6626. 26. Kelly, J.; Snell, M. Hematoporphyrin Derivative: A Possible Aid in the Diagnosis and Therapy of Carcinoma of the Bladder. J. Urol. 1976, 115 (2), 150–151. 27. Zhou, Y.; Liang, X.; Dai, Z. Porphyrin-Loaded Nanoparticles for Cancer Theranostics. Nanoscale 2016, 8 (25), 12394–12405. 28. Ethirajan, M.; Chen, Y. H.; Joshi, P.; Pandey, R. K. The Role of Porphyrin Chemistry in Tumor Imaging and Photodynamic Therapy. Chem. Soc. Rev. 2011, 40 (1), 340–362. 29. Nam, J. S.; Kang, M.-G.; Kang, J.; Park, S.-Y.; Lee, S. J. C.; Kim, H.-T.; Seo, J. K.; Kwon, O.-H.; Lim, M. H.; Rhee, H.-W.; Kwon, T.-H. Endoplasmic Reticulum-Localized


Page 24 of 28

Page 25 of 28

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


Iridium(III) Complexes as Efficient Photodynamic Therapy Agents via Protein Modifications, J. Am. Chem. Soc. 2016, 138 (34), 10968–10977. 30. Shi, H.; Ma, X.; Zhao, Q.; Liu, B.; Qu, Q.; An, Z.; Zhao, Y.; Huang, W. Ultrasmall Phosphorescent Polymer Dots for Ratiometric Oxygen Sensing and Photodynamic Cancer Therapy. Adv. Funct. Mater. 2014, 24 (30), 4823–4830. 31. Lv, W.; Zhang, Z.; Zhang, K. Y.; Yang, H.; Liu, S.; Xu, A.; Guo, S.; Zhao, Q.; Huang, W. A Mitochondria-Targeted Photosensitizer Showing Improved Photodynamic Therapy Effects Under Hypoxia. Angew. Chem. Int. Ed. 2016, 55 (34), 9947–9951. 32. He, L.; Li, Y.; Tan, C.-P.; Ye, R.-R.; Chen, M.-H.; Cao, J.-J.; Ji, L.-N.; Mao, Z.-W. Cyclometalated Iridium(III) Complexes as Lysosome Targeted Photodynamic Anticancer and Real-Time Tracking Agents. Chem. Sci. 2015, 6 (10), 5409–5418. 33. He, L.; Tan, C.-P.; Ye, R.-R.; Zhao, Y.-Z.; Liu, Y.-H.; Zhao, Q.; Ji, L.-N.; Mao, Z.-W. Theranostic Iridium(III) Complexes as One- and Two-Photon Phosphorescent Trackers to Monitor Autophagic Lysosomes. Angew. Chem. Int. Ed. 2014, 53 (45), 12137–12141. 34. Tso, K. K.-S.; Leung, K.-K.; Liu, H.-W.; Lo, K. K.-W. Photoactivatable Cytotoxic Agents Derived from Mitochondria-Targeting Luminescent Iridium(III) Poly(Ethylene Glycol) Complexes Modified with a Nitrobenzyl Linkage. Chem. Commun. 2016, 52 (24), 4557–4560. 35. Lo, K. K.-W.; Tso, K. K.-S. Functionalization of Cyclometalated Iridium(III) Polypyridine Complexes for The Design of Intracellular Sensors, Organelle-Targeting Imaging Reagents, and Metallodrugs. Inorg. Chem. Front. 2015, 2 (6), 510–524. 36. Lo, K. K.-W. Luminescent Rhenium(I) and Iridium(III) Polypyridine Complexes as Biological Probes, Imaging Reagents, and Photocytotoxic Agents. Acc. Chem. Res. 2015, 48 (12), 2985–2995.



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

37. Jiang, Q.; Wang, M.; Yang, L.; Chen, H.; Mao, L. Synergistic Coordination and Hydrogen Bonding Interaction Modulate the Emission of Iridium Complex for Highly Sensitive Glutamine Imaging in Live Cells. Anal. Chem. 2016, 88 (20), 10322–10327. 38. Chen, M.-H.; Wang, F.-X.; Cao, J.-J.; Tan, C.-P.; Ji, L.-N.; Mao, Z.-W. Light-Up Mitophagy in Live Cells with Dual-Functional Theranostic Phosphorescent Iridium(III) Complexes. ACS Appl. Mater. Interfaces 2017, 9 (15), 13304–13314. 39. Yang, Q.; Shi, M.; Zhao, H.; Lin, J.; An, L.; Cui, L.; Yang, H.; Zhou, Z.; Tian, Q.; Yang, S. Water-Soluble Polymer Nanoparticles Constructed by Three Component Self-Assembly: An Efficient Theranostic Agent for Phosphorescent Imaging and Photodynamic Therapy. Chem. Eur. J. 2017, 23 (15), 3728–3734. 40. Liu, Y.; Chen, M.; Cao, T.; Sun, Y.; Li, C.; Liu, Q.; Yang, T.; Yao, L.; Feng, W.; Li, F. A Cyanine-Modified Nanosystem for in Vivo Upconversion Luminescence Bioimaging of Methylmercury. J. Am. Chem. Soc. 2013, 135 (26), 9869–9876. 41. Qiu, K.; Huang, H.; Liu, B.; Liu, Y.; Huang, Z.; Chen, Y.; Ji, L.; Chao, H. Long-Term Lysosomes Tracking with a Water-Soluble Two-Photon Phosphorescent Iridium(III) Complex. ACS Appl. Mater. Interfaces 2016, 8 (20), 12702–12710. 42. Mao, Z.; Wang, M.; Liu, J.; Liu, L.-J.; Lee, S. M.-Y.; Leung, C.-H.; Ma, D.-L. A Long Lifetime Switch-On Iridium(III) Chemosensor for the Visualization of Cysteine in Live Zebrafish. Chem. Commun. 2016, 52 (24), 4450–4453. 43. Fan, N.-C.; Cheng, F.-Y.; Ho, J. A.; Yeh, C.-S. Photocontrolled Targeted Drug Delivery: Photocaged Biologically Active Folic Acid as a Light-Responsive Tumor-Targeting Molecule, Angew. Chem. Int. Ed. 2012, 51 (35), 8806–8810.


Page 26 of 28

Page 27 of 28

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


44. Zhao, Q.; Yu, M.; Shi, L.; Liu, S.; Li, C.; Shi, M.; Zhou, Z.; Huang, C.; Li, F. Cationic Iridium(III) Complexes with Tunable Emission Color as Phosphorescent Dyes for Live Cell Imaging. Organometallics 2010, 29 (5), 1085–1091. 45. Serroni, S.; Juris, A.; Campagna, S.; Venturi, M.; Denti, G.; Balzani, V. Tetranuclear Bimetallic Complexes of Ruthenium, Osmium, Rhodium, and Iridium. Synthesis, Absorption Spectra, Luminescence, and Electrochemical Properties. J. Am. Chem. Soc. 1994, 116 (20), 9086–9091. 46. Zhou, Q.-X.; Lei, W.-H.; Chen, J.-R.; Li, C.; Hou, Y.-J.; Wang, X.-S.; Zhang, B.-W. A New Heteroleptic Ruthenium(II) Polypyridyl Complex with Long-Wavelength Absorption and High Singlet-Oxygen Quantum Yield. Chem. Eur. J. 2010, 16 (10), 3157–3165. 47. Adarsh, N.; Avirah, R. R.; Ramaiah, D. Tuning Photosensitized Singlet Oxygen Generation Efficiency of Novel Aza-BODIPY Dyes. Org. Lett. 2010, 12 (24), 5720–5723. 48. Tian, J.; Ding, L.; Xu, H.-J.; Shen, Z.; Ju, H.; Jia, L.; Bao, L.; Yu, J.-S. Cell-Specific and pH-Activatable Rubyrin-Loaded Nanoparticles for Highly Selective Near-Infrared Photodynamic Therapy against Cancer. J. Am. Chem. Soc. 2013, 135 (50), 18850–18858.



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

Table of Contents

3O 2

GSH

s nucleu

1O 2


Page 28 of 28

Cyclometalated Iridium(III)-Complex-Based Micelles for Glutathione

Recommend Documents