Amphiphilic Gemini Iridium(III) Complex as a Mitochondria-Targeted

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Biological and Medical Applications of Materials and Interfaces

Amphiphilic Gemini Iridium(III) Complex as Mitochondria-Targeted Theranostic Agent for Tumor Imaging and Photodynamic Therapy Sili Yi, Zhen Lu, Jin Zhang, Jun Wang, Zeng-Hong Xie, and Linxi Hou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01205 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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ACS Applied Materials & Interfaces

Amphiphilic Gemini Iridium(III) Complex as Mitochondria-Targeted Theranostic Agent for Tumor Imaging and Photodynamic Therapy Sili Yi,a Zhen Lu,b Jin Zhang, b Jun Wang, a Zenghong Xie,a, * Linxi Houb,* a Institute

of Food Safety and Environment Monitoring of Photocatalysis on Energy and

Environment College of Chemistry, Fuzhou University, Fuzhou 350116, P.R. China. b College

of Chemical Engineering, Fuzhou University, Fuzhou 350116, P.R. China.

KEYWORDS: multifunctional agents, gemini iridium complex, vesicles, imaging, photodynamic therapy.

ABSTRACT: Clinical diagnostic and therapeutic of tumors significantly benefit by the development of multifunctional theranostic agents which integrate tumor targeting, imaging and therapeutics. However, the integration of imaging and therapy functionalities to a unimolecular framework remain a great challenge. Herein, a family of amphiphilic gemini iridium(III) complexes (GIC) Ir1−Ir6 are synthesized and characterized. The presence of quaternary ammonium (QA) groups endows GIC adjustable water solubility and excellent self-assembly properties. Spectroscopic and computational results reveal that introduce QA groups into cyclometalating ligands (C^N ligands) can overcome the drawback of aggregation-caused emission quenching and ensure Ir1−Ir3 with high emission intensity and excellent singlet oxygen (1O2) generation ability in aqueous media. Cell-based assays indicate that Ir3 shows higher cellular uptake efficiency and localizes specifically in the mitochondria, as well as exhibits

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outstanding photostability and impressive phototoxicity index with satisfactory performance in mitochondria targeted imaging and photodynamic therapy (PDT) of tumor cells. Furthermore, in vivo studies further prove that Ir3 possesses excellent anti-tumor activity, and remarkably inhibit the growth of the HepG2 cells under PDT treatment. Consequently, this study presents a promising strategy for designing clinical application potential multifunctional iridium complex theranostic agent for mitochondria targeted imaging and PDT in a single molecular framework.

1. INTRODUCTION Despite tremendous progress in our awareness of tumor, which still one of the most deadliest diseases in the worldwide. As cancer spreads, advanced diagnostic and treatment strategies are essential for early diagnosis and therapy.1−2 Recently, multifunctional theranostic agents are emerging as an substitute to traditional cancer diagnostic tools and therapy strategies.3−4 The integration of therapeutics and diagnostics of cancer in a single procedure, represents a major goal of current biomedical research, which pursues to study therapeutic mechanism, improve therapeutic efficacy and realize personalized cancer therapy.5−6 However, there are still some scientific challenges for the development of multifunctional theranostic agents which combine imaging and therapy functionalities in a single molecular framework until now.7−8 Photosensities (PS) can be used as bioimaging agents due to its excellent photophysical properties. Upon light irradiation, molecular oxygen can effectively quenched the excited state of the PS to produce reactive oxygen species (ROS) by electron transfer or energy transfer, such as hydroxyl radicals (∙OH) and 1O2.9 The generated ROS is considered as the major toxic species causing severe oxidative stress and triggering cell death.10 Hence, the PS can be performed as the appealing candidates for multifunctional theranostic agents that combine imaging and PDT


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functionalities. Up to date, many organic or inorganic PS have been reported for applications in materials science and biology.11 However, most of organic PS generally occur fluorescence aggregation-caused quenching (ACQ) in water owing to hydrophobic property, greatly reduced photoluminescence quantum efficiency and photocytotoxicity, resulting in inferior imaging and PDT efficacy.12 Inorganic nanomaterials are long term cytotoxicity due to the high uptake by the reticuloendothelial system cause their slow and inefficient clearance from the host.13 Therefore, there are few examples of PS as multifunctional theranostic agents with optimum emission features used in clinic.14 Transition metal complexes have distinct advantages that render them as ideal candidates for organic molecules PS. For instance, transition metal complexes can show a series of excited states, and the heavy atom effect is advantageous to fast intersystem crossing (ISC) from singlet to triplet state. The longer lifetimes result from ISC be beneficial to yield 1O2 and superoxide radicals.15 Meanwhile, their relatively long emission lifetimes can largely eliminate the autofluorescence interference that beneficial for biosensing and bioimaging.16−17 Among various transition metal complexes, iridium complex is widely explored as PS attributed to their high photostability, large stokes shifts, impressive 1O2 quantum yield, good biocompatibility, abundant modification potentials and better cell permeability.18−19 However, iridium complex still have some drawbacks that are difficult to satisfy the balance between the imaging and therapeutic desires. Considering the hydrophilic environment of biological systems, the hydrophobic nature of most iridium complex also results in the ACQ that seriously affect the imaging and PDT effect.20−21 The ACQ problem greatly restrict their practical applications. Furthermore, the metal iridium center usually exhibit high cytotoxicity toward eukaryotic cell lines.22 Therefore, developing novel iridium complexes with appropriate solubility, high



luminescence intensity, low dark cytotoxicity and excellent 1O2 generation ability in aqueous media are much more challenging. Surfactants have been widely applied to nanomedicine by means of enhanced permeability and retention (EPR) effect benefit by the excellent self-assembly capability.23 Moreover, surfactants can form characteristic supramolecular aggregates micelles or vesicles which reduce the probability of π-π stacking between fluorescent molecules, benefit to improved the solubility and 1O2 quantum yield in aqueous media.24 Hence, the strategy of integrating surfactants and iridium complex is expected to significantly enhance imaging and PDT effect. Herein, inspired by the structure and properties of gemini surfactants, a family of amphiphilic GIC Ir1−Ir6 are designed and synthesized, in which the QA groups are used as hydrophilic functional group to modify the C^N ligands and auxiliary ligand (N^N ligand) of hydrophobic iridium complex, respectively. The presence of QA groups endows GIC adjustable water solubility and excellent self-assembly properties. Ir1−Ir6 can spontaneously form vesicles by self-assembly in aqueous media on account of the amphiphilic nature, and exhibit two different arrangements of molecules in the aggregation vesicles due to the position of the QA groups, which are greatly affect their photophysical properties. Notably, introducing the QA groups into the 4-position C^N ligands can overcome the drawback of aggregation-caused emission quenching and ensure Ir1−Ir3 with high emission intensity and excellent 1O2 generation ability in aqueous media. Meanwhile, QA groups improve the binding affinity of the GIC with cell membranes which is negatively charged and facilitates cellular internalization. Cell-based assays indicate that Ir3 shows higher cellular uptake efficiency through an energy dependent endocytosis pathway and localizes specifically in mitochondria, as well as exhibits outstanding photostability and impressive phototoxicity index with satisfactory performance in mitochondria targeted imaging and PDT of tumor cells.


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Furthermore, in vivo studies further prove that Ir3 possesses excellent anti-tumor activity, and remarkably inhibit the growth of the HepG2 cells under PDT treatment. 2. RUSULTS AND DISCUSSION 2.1. Synthesis and Characterization. In order to develop novel multifunctional theranostic agent integrating imaging and PDT functionalities in a single molecular framework. A family of GIC amphiphilic (Ir1−Ir6) were designed and successfully synthesized according to the route shown in Scheme 1. We tried to introduce the QA groups into the C^N ligands and N^N ligand of

iridium

complex,

respectively.

The

C^N

ligands:

2-[4-(tetramethylammonium

bromide)phenyl]pyridine (ppy-tmaBr), 2-[4-(trimethylbutylammonium bromide)phenyl] pyridine (ppy-tbaBr) and 2-[4-(trimethyloctylammonium bromide)phenyl]pyridine (ppy-toaBr). N^N ligands:

4,4’-bis(tetramethylammonium

bromide)-2,2’-bipyridine

(bpy-tma2Br2),

4,4’-

bis(trimethylbutylammonium bromide)-2,2’-bipyridine(bpy-tba2Br2) and 4,4’-bis(trimethyloctyl ammonium bromide)-2,2’-bipyridine (bpy-toa2Br2) were synthesized following the method previously reported.25−26 The dinuclear chloro-bridged iridium precursor [Ir(ppy-R)2Cl]2 were prepared by the literature method.27 Complexes were synthesized by refluxing the corresponding [Ir(ppy-R)2Cl]2 and N^N ligands in dichloromethane/methanol (CH2Cl2/CH3OH = 2/1, v/v). The complexes were purifed by recrystallization using suitable solvents. Detailed experimental procedures and corresponding characterization results are summarized in the experimental section. The structures of Ir1−Ir6 were confirmed by 1H NMR,

13C

NMR and electrospray ion

source high resolution mass spectrometry (ESI-HRMS), infrared spectroscopy (IR) and elemental analysis (Supporting Information Figure S1-S13).



Scheme 1. Chemical structure and synthetic routes of Ir1−Ir6.

2.2. Photophysical Properties. The absorption and emission spectras of Ir1−Ir6 (10 μM) were investigated in water (H2O) and acetonitrile (CH3CN) at room temperature (Figure 1a−b), the photophysical properties data of Ir1−Ir6 are summarized in Table 1. Similar to many cationic iridium complexes, Ir1−Ir6 display a intense absorption bands below 300 nm are attributed to the spin-allowed ligand-centered (1LC) π-π* transition for ligands. The moderately intense absorption bands between 330 and 425 nm result from a integration of ligand-centered charge transfer (1LCCT) and the singlet metal-to-ligand charge transfer transition (1MLCT). The low energy absorption bands for Ir1−Ir3 are found at 415 nm, whereas Ir4−Ir6 these are located at 387 nm, this difference probably ascribe to electronic transitions associated with metal center.28−29 Minor energy absorption bands over 425 nm which are attributed to the triplet metalto-ligand charge-transfer transition (3MLCT) and the spin-forbidden ligand-centered transition (3LC).30 Delicate difference in the absorption spectra of Ir1−Ir6 are observed between water and CH3CN.


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Figure 1. (a) UV-vis absorption and emission spectra of Ir1−Ir3 and b) Ir4−Ir6 in H2O and CH3CN. (c) Emission intensity variation curves of Ir1−Ir6 in CH3CN/H2O mixtures with different H2O fractions. (d) Emission spectra of Ir3 and e) Ir5 in CH3CN/H2O mixtures with different H2O fractions. (f) Photobleaching emission intensity of Ir1−Ir6 under continuous irradiation. (λex = 365 nm).

Upon excitation (λex = 365 nm), Ir1−Ir6 exhibit yellow to red emissions. The emission bands are unstructured and broad shape which are typical of pronounced triplet 3MLCT as well as ligand-to-ligand charge transfer transitions (3LLCT) electronic excitations.31−32 The emission for Ir1−Ir3 that the QA groups attach to the 4-position C^N ligands are almost the same with a maximum wavelength around 545 nm in CH3CN and blue shifted of 40 nm compare to [Ir(ppy)2(bpy)]+ (λem = 585 nm).33 Comparison of the photoluminescence spectra indicate that the emission maxima of Ir1−Ir3 exhibit a slightly blue-shifted in water relative to in CH3CN, and have not significantly depend on the length of the alkyl chains. In contrast, the emission maxima for Ir4−Ir6 which the QA groups indwell on the N^N ligand not only with significantly red7 Environment ACS Paragon Plus


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shifted compare to [Ir(ppy)2(bpy)]+, but also show sensitive emission towards solvent and the length of the alkyl chains. These trends among them can be assigned to the electron mobility trends from the ligands to the strong electron withdrawing QA groups, which has a strong effect on the electron density around the center of iridium, thereby resulting in a distinct energy gap. 34

Table 1. Photophysical properties of complexes Ir1−Ir6 in H2O and CH3CN at room temperature. Complex

Ir1

Ir2

Ir3

Ir4

Ir5

Ir6

(a)

λmax.abs [nm]

λmax.em

ΦP

Φs

τ

Kr

Knr

[ε×104 M-1cm-1] a

[nm] b

[%] c

[%] d

[ns] e

[S-1] f

[S-1] f

H2O

256(3.60),304(1.80),415(0.24)

540

12.2

0.42

102.08

1.19×103

8.60×103

CH3CN

257(2.41),302(0.86),418(0.16)

545

4.45

n.dg

4.86

9.25×103

1.96×105

H2O

255(4.12),303(2.23),414(0.28)

540

14.6

0.46

168.79

0.86×103

5.06×103

CH3CN

256(3.62),303(1.62),419(0.20)

545

4.52

n.d

5.72

8.04×103

1.66×105

H2O

255(4.42),302(2.52),414(0.32)

540

16.0

0.54

217.51

0.73×103

3.86×103

CH3CN

257(4.25),303(2.01),420(0.25)

545

4.65

n.d

7.47

6.29×103

1.27×105

H2O

250(3.70),320(1.51),374(0.62)

645

0.25

0.12

3.12

0.80×103

3.19×105

CH3CN

252(3.85),319(1.52),379(0.65)

632

1.42

n.d

6.63

2.21×103

1.48×105

H2O

250(3.46),320(1.45),375(0.56)

660

0.10

0.13

1.37

0.73×103

7.29×105

CH3CN

251(4.15),318(1.70),380(0.70)

633

1.65

n.d

10.33

1.55×103

9.52×104

H2O

251(3.02),321(1.20),374(0.52)

664

0.05

0.12

0.74

0.67×103

1.34×106

CH3CN

252(4.50),319(1.82),380(0.76)

635

1.82

n.d

14.57

1.23×103

6.73×104

Solvent

Absorption maxima (molar extinction coefficient);

(b)

Emission maxima;

(c)

ΦP refers to the

photoluminescence quantum yield, which used [Ru(bpy)3]2+ as standard (ΦP = 0.028 in Water);[27] (d)

Φs was detected in H2O by indirect methods with [Ru(bpy)3]2+ (Φs = 0.18 in H2O) as a

reference;[28] (e) Lifetime data: excitation wavelength 365 nm; (f) kr and knr values in solution were calculated using the equations: kr = Φ/τ and knr = (1 – Φ)/τ; (g) n.d = not determined.


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Meanwhile, the observed emission for Ir1−Ir6 show large stokes shifts, implying minimal interference between the emission and excitation light. The photoluminescence quantum yield (ΦP) of Ir1−Ir3 are higher than Ir4−Ir6 in the same spectral region. Notably, the ΦP and emission intensity of Ir1−Ir3 are significantly enhanced in H2O compare to in CH3CN, while Ir4−Ir6 suffer from emission quenching and show the opposite variation trend. To verify this phenomenon, we studied the emission properties of Ir1−Ir6 (10 μM) using mixed solution with various fractions of H2O and CH3CN, the emission intensity variation curves of Ir1−Ir6 are shown in Figure 1c. Ir1−Ir3 and Ir4−Ir6 also exhibit completely opposite variation trend of emission intensity. Take Ir3 and Ir5 as examples, Ir3 exhibits faint emission in pure CH3CN and the emission intensity is significantly enhanced with the H2O content increases, which is about 4-fold than that in pure CH3CN. In stark contrast, the emission intensity of Ir5 decreases obviously with the increase of H2O fractions, the emission intensity about 0.15-fold is less than that of in pure CH3CN. The luminescence photographs provide a forceful evidence for this phenomenon (Figure 1d−e). Further information about the emission lifetime (τ) of Ir1−Ir6 are obtained by time-resolved phosphorescence measurements. Their emission decay profiles that evidently indicate the lifetimes of Ir1−Ir6 have slightly extended with the increase of the length of the alkyl chain, and have similar lifetimes in CH3CN (Figure S14). However, the variation trend of time-resolved emission decay of Ir1−Ir6 are similar to that of quantum yield in water, which Ir1−Ir3 exhibit a significantly longer lifetimes. From the ΦP and τ values, the radiative decay rates (kr) and nonradiative decay rates (knr) are calculated by the equations kr = Φ/τ and knr = (1 − Φ)/τ.35 Clearly, the kr value of Ir1−Ir6 are relatively invariable, irrelevant to the substituent on the ligands. Whereas the knr value of Ir4−Ir6 are much larger than those of Ir1−Ir3 in water. These higher knr value of Ir4−Ir6 are consistent with the relatively



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shorter emission lifetimes. In general, the nonradiative processes of decay largely control the relative quantum yield of complexes, since the variations of kr relative to a large knr variation range is limited. Meanwhile, the knr value is probably correlate with the arrangement of molecules in the state of aggregation and surrounding medium environment, which might manipulate the nonradiative pathway of decay.36 Therefore, the possible reason that the variation trend both the emission properties and lifetimes of Ir1−Ir6 in water are attribute to the different arrangement of molecules in the aggregation vesicles due to the position of hydrophilic QA groups, and then affect the nonradiative pathway of decay. To investigate the photostability, the emission intensity of the Ir1−Ir6 under continuous irradiation using an UV-lamp (365 nm) were recorded, as shown in Figure 1f. After 1 h of irradiation, the final emission intensity of the Ir1−Ir6 are still maintained above 80%. All of the data indicate that the location of QA groups play a functional role in turning the photophysical performances of GIC. This is a good strategy that introduce QA groups into the 4-position C^N ligands to improve the emission intensity and prolong the lifetimes of the iridium complex in aqueous media, which is considered to be advantageous for bioimaging applications. 2.3. Amphiphilic and Self-Assembly of Vesicles. Considering the hydrophilic environment of pericellular and the cellular membrane negatively potential, appropriate hydrophilicity and potential of PS are vital for cell uptake efficiency which is beneficial to cellular imaging and PDT. The octanol/water partition coefficient (log Po/w) is one of the critical parameters for relative solubility in oil and water, such a coefficient is known to use for prediction of the cell uptake efficiency.39 Herein, the shake-ﬂask method was used to measured the log Po/w values of Ir1−Ir6

according

to

literature

procedure.40

The


experiment

results

indicate

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Figure 2. (a) The octanol/water partition coefficient of Ir1−Ir6. (b) Zeta potential of the Ir1−Ir6 aqueous solution. (c) TEM images of Ir1−Ir6 vesicles. (d) The hydrodynamic diameters of Ir1−Ir6 vesicles in aqueous media. (e) AFM images of Ir3 and f) Ir5 aqueous solution.

that Ir1−Ir6 exhibit amphiphilic characteristic and the hydrophilicity can be adjusted by controlling the length of hydrophobic alkyl chains (Figure 2a). These values can be supported by the observation that the luminescence of the octanol/water mixtures (Figure S15). The zeta potential of the Ir1−Ir6 aqueous solution are 20.9 − 40.8 mV (Figure 2b), clearly suggesting that the longer alkyl chains are conducive to form stable vesicles in water, and the positive charge of QA ions around the surface of vesicles. Adjustable water solubility and potential show that GIC have broad application potential in the field of biomedicine. The self-assembly vesicles morphological of Ir1−Ir6 in water are studied by transmission electron microscopy (TEM) and dynamic light scattering (DLS), as shown in Figure 2c−d. The TEM images show that Ir1−Ir6 self-assemble into aggregates and exhibit a good spherical morphology with a diameter of approximately 12 ± 2 nm, 16 ± 2 nm, 20 ± 2 nm, 26 ± 2 nm, 30 ± 11 Environment ACS Paragon Plus


2 nm, 36 ± 2 nm, respectively. Also, the DLS measurement further indicate that Ir1−Ir6 present a relatively uniform size dispersion with an average hydrodynamic sizes of 20 ± 2 nm, 27 ± 2 nm, 34 ± 2 nm, 30 ± 2 nm, 44 ± 2 nm, 52 ± 2 nm in water, respectively, indicating that these aggregates should be vesicles not micelles, because the diameter of micelles is in the order of 3−7 nm.41−42 It is reasonable that their sizes of the vesicles obtained by TEM are smaller than hydrodynamic diameter, because the hydrodynamic diameter is constituted by the vesicles together with the water coating layer, which is not present for TEM.18,43 Vesicles formation are further confirmed by AFM studies. Take Ir3 and Ir5 as examples because of the similarly log Po/w values, it is clearly observed that Ir3 and Ir5 can spontaneously form uniform spherical vesicles in water (Figure 2e−f). The vesicles of Ir5 have a larger diameter compare to Ir3, which is mainly because the different arrangement of molecules in the aggregated vesicles due to the position of the QA groups. The dispersions of Ir1−Ir6 are highly stable without aggregation and the average hydrodynamic diameters remained almost unchanged even after storage for half months (Figure S16). The high stability of the vesicles probably due to amphiphilic nature as well as strong electrostatic repulsion. Research indicates the aggregation morphology and the arrangement of molecules in the aggregation of functional dyes can greatly affect their electronic absorption and optical properties.44 In the solution phase, the arrangement of molecules in the aggregation depends on the structural features of the dye molecule.45 The vesicles are spontaneously formed by selfassembly in water owe to the amphiphilic nature of Ir1−Ir6. The aggregation process of complex are affected by the hydrophobic attractions between the tail units, electrostatic repulsions between the head groups and π-π attractions between aromatic moieties.46 In the midst of water, the aromatic moieties and two symmetrical alkyl chains as hydrophobic tail groups are arranged


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Scheme 2. Schematic representation of the formation of the aggregated vesicles in aqueous media (Take Ir3 and Ir6 as representatives).

face each other to make up the core of the bilayer, the QA groups as the polar hydrophilic head groups are exposed to the water medium, acting as a shield towards the phosphorescent core. However, Ir1−Ir6 exhibit two different arrangements of molecules in the aggregated vesicles due to the position of the QA groups. Take Ir3 and Ir6 as representatives, the related schematic diagram of the formation of the aggregated vesicles in aqueous media is shown in Scheme 2. In general, the traditional fluorescent dyes are easily occur intermolecular π-π stacking in the aggregation state, the strong π-π stacking interaction cause the intermolecular energy transfer, resulting that the excited state energy was decayed by nonradiative pathway, and then showing



the aggregation state fluorescence quenching.47−48 For Ir3, the dipyridyl groups arrange by an edge-to-edge configuration are not tend to occur strong intermolecular π-π interactions in aggregation vesicles, which largely block the intermolecular nonradiative energy transfer, reducing the nonradiative decays. Meanwhile, these aggregated vesicles create a protecting environment for the 3MLCT excited state, and avoid other non-radiative channels form excimer that consume the excited state energy. As a consequence, the excited-state energy is mainly transformed into photons, resulting in strong emission.49 Whereas the aromatic groups of Ir6 are perpendicular to each other and stagger in an edge-to-face T-type stack configuration, which show intensity π-π stacking interactions and increase the nonradiative energy transfer among the molecules, leading to the decrease of emission intensity.50 This explanation is supported by the comparably lower quantum yield and weaker emission intensity of Ir4−Ir6 with relatively high knr values. 2.4. Electrochemical Properties. Electrochemical measurements can approximate evaluate the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels of the ground state of iridium complex. The redox behavior of complexes Ir1−Ir6 were investigate by cyclic voltammetry. The Cyclic voltammograms for Ir1−Ir6 are shown in Figure S17. The electrochemistry data of Ir1−Ir6 are summarized in Table 2. The first oxidation potential of Ir1−Ir3 are 1.41 V, 1.40 V, and 1.39 V, respectively, which is assigned to Ir-based oxidation (IrIII/IrIV) with contribution from the C^N ligands, and with a significantly anodically shifted relative to Ir4−Ir6. This is mainly due to the electron withdrawing QA groups on the C^N ligands lower the electron density around the iridium center and thus inhibit the oxidation of complexes.51−52 Likewise, Ir4−Ir6 display a higher reduction potentials because of the QA groups attached to the N^N ligands and induced appreciable anodic shift in the reduction


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Table 2. Electrochemical Data of Ir1−Ir6

(a)

Complex

Eox [V] a

Ered [V] a

HOMO [eV] b

LUMO [eV] b

Eg [eV] c

Ir1

1.41

-1.29

-6.15

-3.45

2.70

Ir2

1.40

-1.29

-6.14

-3.45

2.69

Ir3

1.39

-1.30

-6.13

-3.44

2.69

Ir4

0.92

-0.87

-5.66

-3.87

1.79

Ir5

0.93

-0.86

-5.67

-3.88

1.79

Ir6

0.93

-0.88

-5.67

-3.86

1.81

Determined by cyclic voltammograms in deaerated CH3CN, n-NBu4PF6 (0.1 M) was used as

the supporting electrolyte. The redox potentials are quoted versus the ferrocene/ferrocenium: E1/2 = 0.56 V vs Ag/AgCl,53 scan rate = 100 mV s-1.

(b)

HOMO = -e (Eox + 4.74) [eV] ; LUMO = -e

(Ered + 4.74) [eV]; (c) Eg = (Eox - Ered) [eV].

potentials.54 The redox potentials have not significantly affected by length of the alkyl chains. The corresponding oxidation and reduction potentials were used to calculate the HOMO and LUMO energies and the electrochemical energy gaps (Eg). For Ir1−Ir6, the calculated Eg were 2.70 eV, 2.69 eV, 2.69 eV, 1.79 eV, 1.79 eV, and 1.81 eV, respectively. The electron withdrawing QA groups stabilizes the HOMO and LUMO of complexes, thereby resulting in distinct Eg between Ir1−Ir3 and Ir4−Ir6.55 The Eg of Ir4−Ir6 are smaller than those of Ir1−Ir3, which identify with the significantly red-shifted emission from Ir4−Ir6. 2.5. Theoretical Calculations. In order to clearly comprehend the impact of QA groups on the photophysical performances of GIC, the ground and excited electronic states were studied by theoretical calculation which combined density functional theory (DFT) and time dependent density functional theory (TDDFT) using Gaussian 09 program package. The counter ions were 15 Environment ACS Paragon Plus


omitted in the geometry optimizations to simplify the chemical structures of GIC. Preliminary DFT calculations were performed for Ir1−Ir6 at the B3LYP level of theory with the LAN2LDZ basis set for iridium and the 6−31 G* for other atoms. 33,56−57 First, the geometry optimization on the ground state were be performed without symmetry restrictions, Ir1−Ir6 show a approximate octahedral coordination around iridium centre. According to the schematic diagram of the molecular orbital structures and the energy levels of Ir1−Ir6 (Figure 3), the HOMO are reside primarily on both the metal iridium center and aryl ring of the C^N ligands, while the LUMO is largely localized on the N^N ligand. As a result of electron-withdrawing QA groups on the C^N ligands, the HOMOs of Ir1−Ir3 are significantly stabilized result in their HOMO energy levels reduced. Similarly, QA groups are attached to the N^N ligand make for the LUMOs stabilization and lower the LUMO energy levels of Ir4−Ir6. As a consequence, the attachment of electron-withdrawing QA groups play a significant role in adjusting the energy levels. The calculated HOMO−LUMO energy gaps of Ir1−Ir6 are 3.62 eV, 3.56 eV, 3.55 eV, 1.52 eV, 1.59 eV, 1.61 eV, respectively. The calculated results identify with the observed shifted emission. This trend in substituent effect are also in broad accord with the trend of iridium complexes as previously reported.58−59 To further studied the nature of the excited state properties of Ir1−Ir6 with the TDDFT calculation based on the geometry optimization of ground state. The vertical excitation energies involved in the dominant excitations states are summarized in Table S1. The corresponding spin density distributions are shown in Figure S18. Their lowest triplet state (T1) of Ir4−Ir6 mainly assign to transition from HOMO to LUMO and exhibit a mixed 3MLCT and 3LLCT character. However, the T1 of Ir1−Ir3 show a larger multiconfigurational character at the TDDFT level due


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Figure 3. The HOMO and LUMO energy levels and corresponding electron distributions based on the optimization of the ground state for Ir1-Ir6 from DFT calculations.

to their proximity to higher excited states. According to Figure 3, the electron distribution of HOMO-1 is mainly located on the C^N ligands, whereas LUMO+1 is spread over the entire C^N ligands, demonstrating that the emissions of Ir1−Ir3 are predominantly from 3MLCT and C^N ligands charge (LC) transfer. The T1 is generally determines the observed phosphorescence.60 Selected Ir3 and Ir5 as examples, for Ir3 the excitation energies of T1−T4 (2.73 eV, 2.75 eV, 2.78 eV, 3.01 eV) are higher than those of Ir5 (2.06 eV, 2.70 eV, 2.72 eV, 2.78 eV), clearly inferring that triplets are intensely confined on Ir5.61 Such a trend identify with the experimental 17 Environment ACS Paragon Plus


results. However, The difference between the measured value of cyclic voltammetry and the calculated value of DFT is a systematic error, which is due to the fact that the parameters used in DFT calculation are not completely consistent with practicality.62 2.6. Singlet Oxygen and In Vitro Photodynamic Activity. Under light irradiation, the triplet excited state of PS yield ROS.63 To identify the type of ROS produced by Ir1−Ir6, we investigated the electron spin resonance (ESR) spectroscopy used 2,2,6,6-Tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as 1O2 and ∙OH trapping agent, respectively.37 Characteristic 1O2-induced signals are recorded in the air-saturated aqueous solution of Ir1−Ir6 and TEMP under light irradiation. No signals could be detected without light irradiation. However, a characteristic triplet of signals with a relative intensity of 1:1:1 are observed between 3480 and 3530 G after irradiation, and the signal intensity enhanced with the irradiation time increased (Figure 4a), indicating that 1O2 is produced by Ir1−Ir6 under light irradiation. After irradiation of the air-saturated aqueous solution of Ir1−Ir6 and DMPO, no signals could be detected, suggesting that no hydroxyl radicals (∙OH) are formed (Figure S19). To evaluate the 1O2 generation efficiency of Ir1−Ir6, the relative 1O2 quantum yield (Φs) were measured, using 1,3-diphenylisobenzofuran (DPBF) as 1O2 indicator and [Ru(bpy)3]2+ as standard PS under cell free conditions. The air-saturated aqueous solution of Ir1−Ir6 and DPBF were irradiated with solar simulator in a time interval from 0 to 120 s, and the variation of the absorbance for DPBF was monitored at 410 nm (Figure 4b). The plot reveals that the Ir3 with a higher slopes is more efficient 1O2 generators than the others. The efficacy of 1O2 generation of the complexes follow the order: Ir3 ＞ Ir2 ＞ Ir1 ＞ Ir6 ＞ Ir5 ＞ Ir4, which are consistent with their increasing emission lifetimes (Table 1). This can be reasonably explained that more efficient collisional triplet electron transfer and energy transfer from Ir1−Ir3 to molecular oxygen 18 Environment ACS Paragon Plus

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Figure 4. (a) ESR signals of Ir3 trapped by TEMP with times. (b) Rate of decay of DPBF sensitized by Ir1-Ir6 in H2O by the variation of the absorbance at 410 nm. (c) Cell viability of HepG2 and MCF-7 cells incubate with Ir1, (d) Ir2, (e) Ir3, (f) Cisplatin in the dark or light irradiation.

Table 3. Dark- and photo-toxicitya of Ir1−Ir3 towards tumor cells. HepG2 cells Complex

Dark

Light

Ir1

＞300

3.3

Ir2

＞300

Ir3 Cisplatin

MCF-7 cells Phototoxicity

Phototoxicity

Dark

Light

＞90.9

＞300

4.0

＞75.0

2.2

＞136.3

＞300

2.6

＞115.4

＞300

1.2

＞250.0

＞300

1.3

＞230.7

15.6

14.9

1.05

19.8

16

1.24

index

b

index

(a) IC50 values were an average of three measurements; (b) Phototoxicity index (PI), which is the ratio between the IC50 values in the dark upon light irradition



will be allowed by a longer-lived excited state.64−65 The higher Φs values of Ir1−Ir3 demonstrate that they have the potential to use as PDT agents. The potential application of Ir1−Ir3 for photodynamic therapy have been assessed in toward HepG2 and MCF-7 cells as representative tumor cells via 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay with the concentration from 1 μM to 300 μM under dark and irradiation conditions.35 Under dark condition, Ir1−Ir3 exhibit slightly cytotoxicity toward these cells and the half maximal inhibitory concentration (IC50) values over 300 μM (Figure 4c−e and Table 3). The possible reason is that heavy atom iridium is wrapped by the aggregated vesicles which avoid interacting nonspecifically with the proteins in cellular and triggering intracellular immunogenicity and antigenicity.66 The lower dark toxicity is an important characteristic for the PDT agent. However, after incubation the cells with Ir1−Ir3 for 4 h at various concentrations and following irradiated with a xenon lamp (400−700 nm, 50 mW cm-2, 15 min). Ir1−Ir3 show a higher phototoxicity index (PI) that the cell viability decrease rapidly with the increased concentration, indicating that Ir1−Ir3 can efficiently kill tumor cells under light irradiation. In comparison, Ir3 become highly phototoxicity to HepG2 and MCF-7 cells with an IC50 value as low as 1.2 μM (PI = 250.0) and 1.3 μM (PI = 230.7), respectively. Relatively higher PDT efficiency could be attributed to the higher cellular uptake efficiency improved the internalization by cells due to the appropriate lipophilicity and positive charged. Importantly, cisplatin-treated or untreated tumor cells are evaluated to the same irradiation procedure and found not possess any photodynamic response (Figure 4f), revealing that the phototoxicity in intracellular mainly originate from the reaction between the oxygen and excited complexes.


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2.7. Cellular Uptake and Localization. As we all know, the cellular uptake behaviour of metal complex is generally related to many factors, such as lipophilicity, molecular size and substitute groups.67 The cellular imaging and PDT treatment efficacy of PS depend on their cellular uptake. Ir3 was chosen for further studies, considering that its excellent PDT active in tumor cells. The cellular distributions of Ir3 was investigated by co-localization experiment with 𝑅 Red FM (MTR), the the commercially available mitochondrial imaging agent MitoTracker○ 𝑅 Lysosomesnuclear imaging agent Hoechst 33342 and the lysosomal imaging agent Cell Light○

RFP (RFP) in HepG2 cells, as show in Figure 5. The distribution of Ir3 overlapped well with mitochondrial dye MTR, Pearson’s co-localization coefficients value of 0.82. In contrast, a poor

Figure 5. (a) Colocalization images of Ir3 and MTR. (b) Colocalization images of Ir3 and RFP. The excitation wavelengths for Ir3, MTR and MFR are 405 nm, 543 nm and 543 nm, respectively. Emission filter: 520 ± 20 nm (for Ir3), 610 ± 20 nm (for MTR), 580 ± 20 nm (for RFP). Scale bars = 25 μm.



correlation coefficient was observed that use the lysosomal dye RFP or the nuclear dye Hoechst 33342 (Figure S20), proving that Ir3 selective localize into mitochondria. Further cellular uptake mechanisms of Ir3 were confirmed by confocal laser scanning microscopy (CLSM) in the presence of various inhibitors or at low temperatures conditions. The cellular luminescence was not be detected when pre-treatment the tumor cells with metabolic inhibitor and endocytic inhibitor or in the low temperature, indicating that the endocytosis was restrained and Ir3 was untaken by tumor cells. Whereas pre-treatment of the cells with low cell membrane potential and organic cation transporters inhibitor show unsuppressed cellular luminescence, suggesting that Ir3 is uptaken by tumor cells mainly through an energy dependent endocytosis pathway (Figure S21). 2.8. Fluorescence Imaging. The photostability of PS is the crucial for cellular imaging.34 Bright luminescent signals can be observed from Ir3-stained cells, indicating that Ir3 is promising probes for mitochondria targeted imaging. We further investigated the photobleaching properties of Ir3 in living HepG2 cells by continuous laser irradiation. The percentages of the weakened emission signal are calculated by normalized initial emission intensity. The bright luminescent signals from Ir3 are stable and the luminescent intensity remained above 80% of the initial intensity after scanning for 300 s (Figure S22). The images before and after scanning of Ir3 provided strong evidence that Ir3 possesses excellent photostability and can be used as a mitochondria targeted imaging agent application in biology. 2.9. In Vivo PDT Efficacy of Ir3. Preliminary in vitro studies results indicate that Ir3 localizes specifically in the mitochondria and exhibit excellent PDT efficiency. To further confirm the therapeutic capacity of Ir3, the experiments in vivo were carried out using HepG2


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Figure 6. (a) Representative photographs of HepG2 tumor-bearing nude mice before and after different treatments. (b) Tumor volumes curves of four groups after various treatment. (c) Body weight curves of four groups after various treatment. (d) Tumor weights of four groups after 14 days of treatment. (e) H&E stained tumor slices of different groups. Scale bar: 100 μm.

tumor-bearing mice as model. We randomly divided the nude mice into four groups (control group, light group, dark groups, PDT group). Ir3 was administered by intratumoral injection into the tumor bearing mice at a dose of 59.5 μg kg-1 (25 μL, 40 μM) and exposed to a light irradiation (400−700 nm, 250 mW cm-2, 20 min) at 15 min post injection. The same dose and light irradiation were utilized with dark and light groups, respectively. The relative body weight and tumor volumes variation were recorded every 2 days to evaluate the therapeutic effect



Figure 7. Histological examination of normal organs (lung, liver, spleen, kidney and heart) in the PDT group after 14 days of treatment. Scale bar: 100 μm.

(Figure 6a−b). The growth of tumor in PDT group are remarkably inhibited and the volumes slightly decreases as time pass away. In marked contrast, the tumor volumes of the other control groups are increased dramatically in the same period. The body weights are not significantly


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change are observed for all groups after 14 days (Figure 6c). After the treatments, the mice were executed and then collected the tumors and normal organs (heart, liver, spleen, lung, and kidney) after 14 days of treatment for the possible pathomorphology analysis. The tumor weight is much smaller in the PDT group compared to other groups (Figure 6d). Hematoxylin and eosin (H&E) staining of tumor slices indicate that the architectures of tumors are severely damaged, along with significantly reduced cell density in the tumors of the PDT group, and the other control groups show densely packed neoplastic cells (Figure 6e). The normal organs of all groups were also stained by H&E, the microscopy images of the stained slices are shown in Figure 7. There are little indication of noticeable pathological abnormalities or inflammatory lesion, cell necrosis, or apoptosis in organs are found in the organ on H&E staining. Therefore, all the experiments indicated that Ir3 are nontoxic to normal organs and can serve as efficient PDT agent for tumor therapy. 3. CONCLUSIONS In summary, a family of amphiphilic GIC Ir1−Ir6 have been successfully developed. The presence of the QA groups endow GIC adjustable water solubility and excellent self-assembly properties. Ir1−Ir6 can spontaneously form vesicles by self-assembly in aqueous media on account of amphiphilic nature, and exhibit two different arrangements of molecules in the aggregated vesicles. We have unexpectedly found that introduce QA groups into the C^N ligands overcome the drawback of aggregation-caused emission quenching and ensure Ir1−Ir3 with high emission intensity and excellent 1O2 generation ability in aqueous media due to the dipyridyl groups arrange by an edge-to-edge configuration and inhibit the intermolecular π-π stacking in aggregation vesicles. These aggregated vesicles create a protecting environment for the 3MLCT excited state, and avoid other non-radiative channels form excimer that consume the excited state



energy. With appropriate water solubility and positive charged, Ir3 shows higher cellular uptake efficiency by an energy dependent endocytosis pathway and localizes specifically in the mitochondria, as well as exhibits excellent photostability, low dark cytotoxicity and impressive phototoxicity index with satisfactory performance in mitochondria targeted imaging and PDT of tumor cells. Furthermore, in vivo studies further proven that Ir3 possess excellent anti-tumor activity, and remarkably inhibit the growth of the tumors under PDT treatment. Additional, high water solubility is helpful to improve its renal clearance efficiency from body, although the renal clearance behavior for this family of GIC has to be clarified in further studies. Therefore, this work presents a promising strategy for designing clinical application potential multifunctional iridium complex theranostic agent towards mitochondria targeted imaging and PDT in a single molecular framework. 4. EXPERIMENTAL SECTION 4.1. Materials and Measurements. All reagents and solvents are analytical grade and purchased from Aladdin Ltd. (Shanghai, China) unless otherwise indicated. Iridium chloride hydrate is an industrial product (Alfa Aesar). 1H

NMR and

13C

NMR spectra were performed on a Bruker Avance III 500 MHz

spectrometer with MeOD as the solvent. Elemental analysis were recorded on Germany Elemental Vario EL cube analyzer. High resolution mass spectra were obtained using a Agilent 6500 QTOF-MS spectormeter. Fourier transform infrared (FT-IR) spectroscopy was conducted on a Nicolet iS50 FTIR spectrometer. Photophysical data for Ir1−Ir6 (10 μM) were obtained in H2O and CH3CN at room temperature; UV-vis absorption spectra were measured on Thermo Fisher G10S spectrophotometer; Photoluminescence spectra were investigated on Hitachi


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F−4600 spectrometer. Emission decays measurements were collected using Horiba DeltaProTM instrument. The morphology of the vesicles were measured by Tecnai F20 transmission electron microscopy (200 kV). The particle sizes and zeta potential of the vesicles were measured by Malvern Zetasizer NanoZS-E with back scattering angle of 137° at room temperature. AFM measurements were taken with a Oxford Cypher S AFM Microscope. In AFM measurements, the sample (10 μM, 50 μL) was dropped onto the mica and make sure the solution is tiled on the mica. Whereafter, AFM was performed with a tapping mode, the scan speed of 3-5 Hz. Cyclic voltammetry was carried out on the CHI660 electrochemical analyzer with a scan rate of 100 mV s-1 in nitrogen-purged CH3CN solutions. Tetrabutylammonium perchlorate (0.1 M) in CH3CN was used as the supporting electrolyte with ferrocene as the internal standard. A glass carbon electrode, Ag/AgCl electrode and platinum wire were used as working electrode, reference electrode and counter electrode, respectively. 4.2. Synthesis and Characterization. The ligands were synthesized according to pervious work with minor modified.26 In briefly, the C^N ligands and N^N ligands were obtained by quaternization with 2-[4-(Bromomethyl)phenyl]pyridine and 5,5'-Dibromomethyl-2,2'-bipyridine as described in previous work, respectively.25 The dimeric iridium [Ir(ppy-R)2Cl]2 were prepared according to the standard procedure.58 [Ir(ppy-tmaBr)2(bpy)]+Cl- (Ir1): ppy-tmaBr (0.986 g, 3.22 mmol) and IrCl3·3H2O (0.515 g, 1.46 mmol) were dissolved in 2-ethoxyethanol (30 mL) and H2O (10 mL) at a flask. The mixture was refluxed with magnetic stirring under nitrogen atmosphere for 24 h at 125 oC, and then naturally cooled to room temperature. The dinuclear chloro-bridged iridium precursor [Ir(ppytmaBr)2Cl]2 was obtained by evaporated under reduced pressure and washed with ether. A suspension of [Ir(ppy-tmaBr)2Cl]2 (0.168 g, 0.1 mmol) and 2,2'-Bipyridine (0.031g, 0.2 mmol)



were dissolved in CH2Cl2 (20 mL) and CH3OH (10 mL) was refluxed under an nitrogen atmosphere for 16 h. After reaction was finished, the solution was naturally cooled to room temperature and then evaporated to dryness. The crude product was recrystallized from deionized water in low temperature to yield Ir1 as a yellow solid (0.136 g, 73% yield). 1H NMR (500 MHz, MeOD, δ): 9.15 (s, 2H), 8.20−8.14 (m, 4H), 7.91−7.85 (m, 6H), 7.81−7.75 (m, 2H), 7.16−7.10 (m, 2H), 7.05 (d, J = 7.4 Hz, 2H), 6.94−6.90 (m, 2H), 6.34−6.27 (m, 2H), 4.83 (d, J = 3.2 Hz, 4H), 3.33 (s, 18H). 13C NMR (126 MHz, MeOD, δ): 164.6, 160.6, 156.2, 150.3, 148.3, 146.6, 146.2, 140.0, 135.8, 133.2, 128.1, 127.3, 127.0, 126.5, 126.2, 125.3, 124.6, 123.2, 123.1, 122.8, 122.6, 120.8, 61.3, 57.5, 50.2, 46.6, 39.2, 36.8, 30.8, 27.3, 26.0, 25.1, 24.4, 23.2, 21.5, 20.2, 16.8; Anal. Calcd. for C40H44Br2ClIrN6: C 48.22, H 4.45, N 8.44. Found: C 48.02, H 4.39, N 8.73. [Ir(ppy-tbaBr)2(bpy)]+Cl- (Ir2): The synthesis of Ir2 was similar to that of Ir1 except that the C^N ligand ppy-tmaBr was replaced by ppy-tbaBr. Ir2 was obtained as a yellow solid (0.125 g, 65% yield). 1H NMR (500 MHz, MeOD, δ): 9.49 (s, 2H), 8.2−8.13 (m, 4H), 7.93−7.84 (m, 6H), 7.81−7.75 (m, 2H), 7.18−7.12 (m, 2H), 7.10−7.03 (m, 2H), 6.96−6.90 (m, 2H), 6.33−6.26 (m, 2H), 4.81 (d, J = 6.2 Hz, 4H), 3.36−3.22 (m, 16H), 1.99−1.84 (m, 4H), 1.52−1.42 (m, 4H), 1.10−1.01 (m, 6H).

13C

NMR (126 MHz, MeOD, δ): 164.6, 160.6, 156.2, 150.3, 148.3, 146.6,

146.2, 140.0, 135.8, 133.2, 128.1, 127.3, 127.0, 126.5, 126.2, 125.3, 124.6, 123.2, 123.1, 122.8, 122.6, 120.8, 61.3, 57.5, 50.2, 46.6, 39.2, 36.8, 30.8, 27.3, 26.0, 25.1, 24.6, 24.4, 23.2, 22.3, 21.5, 21.0, 20.2, 16.8; Anal. Calcd. for C46H56Br2ClIrN6: C 51.14, H 5.22, N 7.78. Found: C 49.96, H 5.09, N 7.86. [Ir(ppy-toaBr)2(bpy)]+Cl- (Ir3): The synthesis of Ir3 was similar to that of Ir1 except that the C^N ligand ppy-tmaBr was replaced by ppy-toaBr, Ir3 was obtained as a yellow solid (0.138


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g, 70% yield). 1H NMR (500 MHz, MeOD, δ): 9.52 (s, 2H), 8.24−8.11 (m, 4H), 7.99−7.83 (m, 6H), 7.79 (d, J = 5.6 Hz, 2H), 7.19−7.03 (m, 4H), 7.00−6.88 (m, 2H), 6.36−6.25 (m, 2H), 4.81 (d, J = 7.8 Hz, 4H), 3.36−3.25 (m, 16H), 1.96 (s, 4H), 1.60−1.13 (m, 20H), 1.02−0.80 (m, 6H). 13C

NMR (126 MHz, MeOD, δ): 164.6, 160.6, 156.2, 150.3, 148.3 ,146.6, 146.2, 140.0, 135.8,

133.2, 128.1, 127.3, 127.0, 126.5, 126.2, 125.3, 124.6, 123.2, 123.1, 122.8, 122.6, 120.8, 61.3, 57.5, 50.2, 46.6, 39.2, 36.8, 30.8, 29.3, 27.3, 26.0, 25.1, 24.6, 24.4, 23.6, 23.2, 22.3, 21.5, 21.0, 20.2, 16.8; Anal. Calcd. for C54H72Br2ClIrN6: C 54.38, H 6.09, N 7.05. Found: C 54.29, H 5.96, N 7.16. [Ir(ppy)2(bpy-tma2Br2)]+Cl- (Ir4): 2-Phenylpyridine (3.22 mmol) and IrCl3·3H2O (1.46 mmol) were dissolved in 2-ethoxyethanol(30 mL) and H2O (10 mL) at a flask. was refluxed with magnetic stirring under nitrogen atmosphere for 24 h at 125 oC and then naturally cooled to room temperature. The dinuclear chloro-bridged iridium precursor [Ir(ppy)2Cl]2 was obtained by evaporated and washed with deionized water and ether. A suspension of [Ir(ppy)2Cl]2 (0.107 g, 0.1 mmol) and bpy-tma2Br2 (0.092 g, 0.2 mmol) were dissolved in CH2Cl2 (20 mL) and CH3OH (10 mL) was refluxed under an nitrogen atmosphere for 16 h. After reaction was finished, the solution was naturally cooled to room temperature and then evaporated to dryness. The crude product was recrystallized from deionized water in low temperature to yield Ir4 as a red solid (0.148 g, 76% yield). 1H NMR (500 MHz, MeOD, δ): 8.80 (d, J = 8.2 Hz, 3H), 8.31 (d, J = 8.1 Hz, 2H), 8.28−8.25 (m, 2H), 8.15 (s, 3H), 8.04−8.01 (m, 4H), 7.64 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 7.7 Hz, 2H), 7.29−7.25 (m, 2H), 7.25−7.21 (m, 2H), 6.38 (s, 3H), 4.36−4.30 (m, 4H), 3.20 (d, J = 4.0 Hz, 18H). 13C NMR (126 MHz, MeOD, δ): 166.2, 162.6, 158.0, 150.2 ,149.1, 147.6, 141.5, 137.8, 135.2, 130.1, 129.3, 129.0, 128.6, 128.2, 125.6, 124.3, 122.6, 122.0, 120.8, 120.4, 62.3,



58.8, 50.6, 48.6, 38.2, 35.6, 29.8, 28.6, 26.6, 25.2, 23.2, 22.0, 21.2, 20.6, 16.8; Anal. Calcd. for C40H44Br2ClIrN6: C 48.22, H 4.45 N 8.44. Found: C 48.83, H 4.36, N 8.55. [Ir(ppy)2(bpy-tba2Br2)]+Cl- (Ir5): The synthesis of Ir5 was similar to that of Ir4 except that the N^N ligand bpy-tma2Br2 was replaced by bpy-tba2Br2. Ir5 was obtained as a red solid (0.157 g, 75% yield). 1H NMR (500 MHz, MeOD, δ): 8.80 (d, J = 8.2 Hz, 2H), 8.32 (d, J = 8.1 Hz, 2H), 8.29−8.23 (m, 2H), 8.10 (d, J = 4.6 Hz, 2H), 8.06−7.99 (m, 4H), 7.75 (d, J = 5.2 Hz, 2H), 7.67−7.62 (m, 2H), 7.30−7.26 (m, 2H), 7.25−7.21 (m, 2H), 6.41 (d, J = 1.6 Hz, 2H), 4.39−4.26 (m, 4H), 3.37 (s, 2H), 3.33 (s, 10H), 3.12 (d, 4H), 1.78−1.65 (m, 4H), 1.35−1.26 (m, 4H), 0.99 (t, J = 7.4 Hz, 6H).

13C

NMR (126 MHz, MeOD, δ): 166.2, 162.6, 158.0, 150.2 ,149.1, 147.6,

141.5, 137.8, 135.2, 130.1, 129.3, 129.0, 128.6, 128.2, 125.6, 124.3, 122.6, 122.0, 120.8, 120.4, 62.3, 58.8, 50.6, 48.6, 38.2, 35.6, 29.8, 28.6, 26.6, 25.2, 23.2, 22.0, 21.2, 20.6, 16.8; Anal. Calcd. for C46H56Br2ClIrN6: C 51.14, H 5.22, N 7.78. Found: C 50.96, H 5.16, N 7.90. [Ir(ppy)2(bpy-toa2Br2)]+Cl- (Ir6): The synthesis of Ir6 was similar to that of Ir4 except that the N^N ligand bpy-tma2Br2 was replaced by bpy-toa2Br2, Ir6 was obtained as a red solid (0.136 g, 68% yield). 1H NMR (500 MHz, MeOD, δ): 8.80 (d, J = 8.2 Hz, 2H), 8.80 (d, J = 8.2 Hz, 2H), 8.28 (t, J = 9.4 Hz, 4H), 8.11 (d, J = 4.6 Hz, 2H), 8.06−7.95 (m, 4H), 7.74 (d, J = 5.2 Hz, 2H), 7.27 (m, J = 8.0, 1.6 Hz, 2H), 7.25−7.18 (m, 2H), 6.40 (d, J = 1.5 Hz, 2H), 4.44−4.19 (m, 4H), 3.37 (s, 2H), 3.33 (s, 10H), 3.19−3.12 (m, 4H), 1.81−1.66 (m, 4H), 1.41−1.24 (m, 20H), 0.93 (t, J = 7.2 Hz, 6H). 13C NMR (126 MHz, MeOD, δ): 166.2, 162.6, 158.0, 150.2 ,149.1, 147.6, 141.5, 137.8, 135.2, 130.1, 129.3, 129.0, 128.6, 128.2, 125.6, 124.3, 122.6, 122.0, 120.8, 120.4, 62.3, 58.8, 50.6, 48.6, 38.2, 35.6, 29.8, 28.6, 26.6, 24.6, 25.2, 23.2, 23.0, 22.2, 21.2, 20.6, 16.8; Anal. Calcd. for C54H72Br2ClIrN6: C 54.38, H 6.09, N 7.05. Found: C 54.30, H 6.01, N 7.14.


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4.3. Log Po/w measurement.68 The n-octanol/water partition coefficients for Ir1−Ir6 were determined using the flask-shaking method. First, the mixture of water (50 mL) and n-octanol (50 mL) was left shaking for 48 h under room temperature. The n-octanol phase and water phase were used for preparing the complex standard solutions (10 μM). Second, the detection solutions (10 μM) were prapared using the mixed solvents containing n-octanol (25 mL) and water (25 mL) and shaken for 48 h. Third, using ultraviolet spectrophotometry to determined the concentration of complexes in the water phase (Cw) and organic phase (Co) of the detecting solution. The logPo/w is calculated by the following equation (1):

( )

𝑙𝑜𝑔 𝑃𝑜/𝑤 = 𝑙𝑜𝑔

𝐶𝑜

(1)

𝐶𝑤

4.4. Electron spin resonance (ESR) assay. The ESR measurements were carried out on Bruker Model A300 spectrometer. The spin adducts of complexes were detected using three settings as follows: 1 G field modulation, 20 mW microwave power, and 100 G scan range. The spin traps TEMP (20 mM) for trapping 1O2 and DMPO (450 μM) for trapping ∙OOH or ∙OH. The ESR signals of the Ir1−Ir6 (10 μM) before and after light irradiation (400−700 nm, 50 mW cm-2) were recorded. 4.5. Singlet oxygen quantum yield (Φs). The relative Φs values of Ir1−Ir6 were measured by the standard method using 1,3-diphenylisobenzofuran (DPBF) as

1O

2

indicator and

[Ru(bpy)3]2+ (Φs = 0.18) as standard in water. The air-saturated aqueous solution of Ir1−Ir6 (10 μM) containing DPBF (50 mM) was measured under irradiated with solar simulator for which light intensity is about 40% of 100 mW cm-2 and dark conditions in a time interval from 0 to 120 s. The absorption for DPBF at 410 nm was recorded by UV-Vis spectrophotometer. The Φs



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values were calculated by means of the variation of absorption spectral of DPBF at 410 nm with the following equation (2):

∅𝑠(𝐼𝑟) = ∅𝑠(𝑠𝑡𝑑) ×

( )×( ) 𝑆𝐼𝑟

𝐹𝑠𝑡𝑑

𝑆𝑠𝑡𝑑

𝐹𝐼𝑟

(2)

Ir designate the Ir1−Ir6, std designate [Ru(bpy)3]2+. S represents the slope of the plot of the absorbance of DPBF. F stands for the correction factor of absorption, which equals to 1−10−OD (OD is the optical density of complexes and [Ru(bpy)3]2+). 4.6. Cell culture. The HepG2 cells and MCF-7 cells were obtained from Fuzhou university (China). The cells were grown in Dulbecco's modified Eagle’s medium (DMEM) supplemented with streptomycin (100 mg mL-1), 1% penicillin and 10% fetal bovine serum (FBS) at 37 oC under 5% CO2 atmosphere in a humid incubator. The cell culture medium was replacement every 48 h. 4.7. Cell viability assay. The Cell viability was evaluated by MTT assay. HepG2 and MCF7 cells were seeded into a 96-well plate and then culture with Ir1−Ir6 in various concentrations (1, 5, 10, 25, 50, 100, 300 μM) at 37 oC, respectively. For phototoxicity studies, after incubation the cells with Ir1−Ir6 for 4 h, removed the culture medium and washed the cells with phosphatebuffered saline (PBS) 3 times, and added the fresh PBS to wells. Took the cells exposed to the light (400−700 nm, 50 mW cm-2, 15 min). The cells were incubated with DMEM/10% FBS for an another 20 h after irradiation. The un-irradiated cells remained in the dark all the time. Subsequently, the fresh culture medium containing MTT (5 mg mL-1) was added into each well after removed the culture medium and then cultured for additional 4 h at 37 °C, Each well were filled with 100 μL DMSO to dissolve the formazan after the medium was removed. The


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absorbance values of such DMSO solution at 490 nm were be recorded with a microplate reader immediately. 4.8. Cellular localization assay. HepG2 cells were cultured with Ir3 (5.0 μM) at 37 °C for 𝑅 Red FM (150 nM, MTR) or Cell Light○ 𝑅 1 h and then further co-incubated with MitoTracker○

Lysosomes-RFP (150 nM, RFP) or Hoechst 33342 (10 g mL-1) for additional 30 min. Cells were washed with ice-cold PBS 3 times and visualized by Germany. Carl Zeiss. LSM 710 laser confocal microscopy immediately. The excitation wavelengths for Ir3, MTR and MFR are 405nm, 543 nm and 543 nm, resperctively. Emission filter: 520 ± 20 nm (for Ir3), 610 ± 20 nm (for MTR), 580 ± 20 nm (RFP). Scale bars = 25 μm. 4.9. Cell Uptake Mechanism.69 HepG2 cells were seeded into 96-well plate for low temperature and normalized incubation, HepG2 cells were cultured with Ir3 (5.0 μM) at 37 °C and 4 °C for 4 h, respectively. Pre-treating with metabolic inhibition (50 mM 2-deoxy-D-glucose and 5 µM oligomycin) or endocytic inhibition (50 μM chloroquine) or modulation of Membrane Potential (50 µM valinomycin) at 37 °C for 1 h or Cation Transporter Inhibition (1 mM tetraethylammonium) at 37 °C for 30 min. The cells were then washed with PBS and incubated solely with Ir3 (5.0 μM) at 37 °C for 1 h, removed the culture medium, and then washed the cells with ice-cold PBS 3 times and visualize by CLSM immediately. 4.10. In Vivo PDT. Animal experiments were executed according to the protocol approved by the Institutional Animal Care and Use Committee of Fuzhou University. HepG2 tumorbearing mice were obtained from Shanghai SLAC laboratory Animal Co., Ltd. We randomly divided the nude mice into four groups. control group: mice without any treatment, light group: mice were irradiated with light only, dark group: mice were injected with Ir3 and without light;



PDT group: mice were injected with Ir3 and exposed to light. For in vivo tumor therapy, tumorbearing mice were prepared and Ir3 was administered by intratumoral injection into the tumor bearing mice at a dose of 59.5 μg kg-1 (25 μL, 40 μM), and then the tumor site were irradiated by a xenon lamp (400−700 nm, 250 mW cm-2, 20 min) at 15 min post injection. The body weights and tumor volumes were recorded every 2 days. The tumor volume was calculated according to the equation: tumor volume = (tumor length) × (tumor width)2/2. According to agency guidelines and the standard protocol, the mice were executed and then collected the tumors and normal organs (heart, liver, spleen, lung, and kidney) after 14 days of treatment, and the tissue slice were made and then stained with H&E. ASSOCIATED CONTENT Supporting Information. 1H

NMR spectrum, ESI-MS spectrum, FTIR spectrum, Time-resolved photoluminescence

spectra, the octanol /water distribution diagram, the hydrodynamic diameters, spin density images, ESR signals curves, colocalization images, cellular uptake mechanism, images of Ir3 in vitro and triplet excited states data. This information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * Email: [email protected] (L. -X. H). * E-mail: [email protected] (Z. -H. X). ORCID 34 Environment ACS Paragon Plus

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Linxi Hou: 0000-0002-7962-0936. Author Contributions S.L.Y. and Z.L. contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Grant No. 21676057), the Fourth Health Education Joint Development Project of Fujian Province (Grant No. WKJ2016-2-04). We also appreciate the Shenzhen Huasuan Technology Company Limited for the theoretical calculation. REFERENCES (1) Meng, X.; Yang, Y.; Zhou, L.; Zhang, L.; Lv, Y.; Li, S.; Wu, Y.; Zheng, M.; Li, W.; Gao, G.; Deng, G.; Jiang, T.; Ni, D.; Gong, P.; Cai, L. Dual-Responsive Molecular Probe for Tumor Targeted Imaging and Photodynamic Therapy. Theranostics 2017, 7, 1781−1794. (2) Lv, Z.; Wei, H.; Li, Q.; Su, X.; Liu, S.; Zhang, K. Y.; Lv, W.; Zhao, Q.; Li, X.; Huang, W. Achieving efficient photodynamic therapy under both normoxia and hypoxia using cyclometalated Ru(II) photosensitizer through type I photochemical process. Chem. Sci. 2018, 9, 502−512. (3) Li, Y.; Tan, C.-P.; Zhang, W.; He, L.; Ji, L.-N.; Mao, Z.-W. Phosphorescent iridium(III)-bisN-heterocyclic carbene complexes as mitochondria-targeted theranostic and photodynamic anticancer agents. Biomaterials. 2015, 39, 95−104. (4) Lim, E.-K.; Kim, T.; Paik, S.; Haam, S.; Huh, Y.-M.; Lee, K. Nanomaterials for



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Table of Contents Graphic


Amphiphilic Gemini Iridium(III) Complex as a Mitochondria-Targeted

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