Activated Type I and Type II Process for Two-Photon Promoted ROS Generation: The Coordinated Zn Matters

Generation: The Coordinated Zn Matters

Article

Activated Type I and Type II Process for Two-Photon Promoted ROS

Bo Ni, Hongzhi Cao, Chengkai Zhang, Shengli Li, Qiong Zhang, Xiaohe Tian, Dandan Li,*

Jieying Wu, and Yupeng Tian *

Cite This: https://dx.doi.org/10.1021/acs.inorgchem.0c02030

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sı Supporting Information

ABSTRACT: The construction of novel classes of photosensitizers

for eﬃcient reactive oxygen species (ROS) generation is of great

interest, yet it is a challenge. In this work, a bis(terpyridine)zinc(II)

complex (namely, ZnL1) with two-photon absorption activity as an

eﬃcient ROS photogenerator was synthesized. Beneﬁting from the

coordinated Zn, the decreased singlet−triplet energy gap favors the

intersystem crossing process facilitating the singlet oxygen ( O )

generation via energy transfer. In addition, it makes the superoxide

·−

radical (O₂) generation easier. This is an extremely rare study on

two-photon excited ROS generation by activating type I and type II

processes based on a cheaper and bioaccessible Zn complex.

INTRODUCTION

dynamic therapy due to the simple preparation and their

desirable photophysical properties, such as the well-studied

■

Photodynamic therapy (PDT) is a novel cancer treatment

method. It involves three important components, including

photosensitizers (PSs), light, and oxygen. After irradiation,

excited PSs interact with surrounding biomolecules or oxygen

giving reactive oxygen species (ROS) to achieve the purpose of

tumor treatment due to their key roles in cell signal

transduction, cell multiplication, and homeostasis at a low

III

12−15

Ru , Os , and Ir transition-metal complexes.

Mean-

while, the involved metal ions in these complexes are rare,

which has always been a hurdle in cancer-targeting treatment

due to their potential toxicity. Alternatively, the employed PS

bearing heavy atoms can favor the intersystem crossing (ISC)

process and boost the O generation for potential PDT, which

level. Typically, ROS are considered to be related to various

pathological conditions and consist of the hydroxyl radical

will involve disturbing dark toxicity as well. In view of the

specimen photodamage from traditional PSs, the newly

emerging two-photon excited PDT provides an interesting

alternative due to their near-infrared excitation source giving a

(

OH·), hydrogen peroxide (H O ), the superoxide radical

2 2

−

3−5

O₂), and singlet oxygen ( O ).

From these, the

−

^s^u^p^e^r^o^xⁱ^d^e^r^a^dⁱ^c^a^l⁽^O2 ) and singlet oxygen ( O ) are the

main and highly toxic reactive oxygen species, which are

17,18

larger penetration depth and weaker phototoxicity.

Considering the above, we reasonably designed a D-A

conﬁguration bis(terpyridine)zinc(II) complex (ZnL1) with-

out heavy atoms to fabricate a two-photon active material

(Scheme 1). The results revealed that ZnL1 exhibited

considerable two-photon absorption activity in the near-

infrared region. Of note, thanks to the coordination of Zn,

its type I and type II processes for ROS generation has been

activated, thus holding promise as a new strategy for eﬃcient

two-photon PDT.

produced via electron transfer (type I mechanism) and energy

transfer (type II mechanism) processes, respectively. In

particular, the search for photosensitizers (PSs) to achieve

enhanced ROS production via both type I and type II

processes is of pivotal interest.

To date, mostly reported PSs are organic molecules₇,

including porphyrin, phthalocyanine, and their derivatives.

However, these molecules often have some inherent defects;

one defect is that these materials are complex to synthesize and

diﬃcult to purify. In addition, most of them have a high self-

aggregation tendency under physiological environments,

leading to the decrease in ﬂuorescence intensity. Also, their

excitation wavelengths are mostly in the visible region; the

damage to organisms is inevitable, which limits their utilization

Received: July 9, 2020

−10

in living cells and tissue.

To address the above limitations,

the use of metal complexes provides a promising platform for

eﬃcient ROS production and for the accompanied photo-

XXXX American Chemical Society

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Scheme 1. (a) Synthesis Route for New Complexes and (b) Schematic illustration of the Apoptosis Process Induced by

Enhancement Type I and Type II Processes

2.18, 19.93, 14.00. ESI−MS: calculated for [M], 996.30; found,

EXPERIMENTAL SECTION

■

− 2+

36.25. [M-2NO ] /2.

Materials and Apparatuses. All chemicals for sample fabrication

were purchased from Aladdin Industrial Corporation and puriﬁed by

conventional methods. The apparatuses for sample characterization,

single crystal structure, photophysical, and bioimaging relevant

measurements in this work have been put into the Supporting

Information in detail.

RESULTS AND DISCUSSION

Crystal Structures. Herein, the designed molecules with

■

D-A conﬁguration (Figure 1 a) have been constructed, and the

Computational Details. All calculations were recorded using the

Gaussian 09 program, and the details for geometry optimization and

relevant calculations were demonstrated in the Supporting Informa-

tion .

Synthesis and Characterization. Synthesis of L1: To a solution

of N1 (1.0 g, 0.005 mol) in ethanol (60 mL) was added 2-

acetylpyridine (1.22 g, 0.011 mol), KOH (1.17 g, 0.020 mol), and

aqueous ammonia (60 mL, 30%). The reaction mixture was kept at 80

C for 6 h. The precipitate formed after cooling and was collected by

ﬁltration. A yellow crystalline solid L1 was obtained from

recrystallization using ethanol. Yield, 64%. The single crystal of L1

was grown from ethanol/dichloromethane solution. H NMR (400

MHz, d -acetone, ppm): δ 8.99−8.82 (m, 6H), 8.19 (m, 1H), 8.12 (s,

H), 8.03−7.90 (m, 2H), 7.62 (m, 1H), 7.52−7.36 (m, 2H), 7.35−

.16 (m, 2H), 4.50−4.28 (m, 2H), 1.53−1.33 (m, 2H), 1.20 (t, J =

.1 Hz, 2H), 1.03−0.90 (m, 3H). C NMR (100 MHz, d -DMSO): δ

(

ppm) 155.39, 155.30, 149.27, 145.11, 137.33, 136.98, 129.60,

Figure 1. (a) Structural formulas of L1 and ZnL1. (b) Crystal

structures of L1 and ZnL1; the displacement ellipsoids are drawn at

the 30% probability level (for clarity, nitrate ions and H atoms within

25.14, 124.29, 122.06, 120.80, 120.74, 119.20, 116.86, 112.16,

10.97, 45.56, 40.13, 39.93, 39.72, 39.51, 39.30, 39.09, 38.88, 31.74,

9.50, 13.53. ESI−MS: calculated for [M + H] , 405.20; found,

05.20.

the molecules have been omitted). (c) The calculated ΔE of L1 and

ZnL1.

Synthesis of ZnL1: The ligand L1 (0.4 g, 1.0 mmol) and

Zn(NO ) ·6H O (0.18 g, 0.7 mmol) were dissolved in ethanol (50

mL), and the solution was heated under reﬂux with stirring for 3 h.

Most of the ethanol was evaporated and cooled to room temperature,

and red microcrystals are gradually precipitated. ZnL1 was collected

by ﬁltration and washed with methanol. Yield, 68%. The single crystal

synthetic procedures were illustrated in Scheme S1. Figures

S1 −S9 displayed the detailed characterization data for

intermediate and target products. The structural features of

L1 and ZnL1 were analyzed by single crystal X-ray diﬀraction

and shown in Figure 1b and Figure S10. Crystal structure

shows that the C−C bonds between the indole ring and the

electron acceptor (pyridine ring) of L1 and ZnL1 are 1.471

and 1.462 Å (L1 > ZnL1), respectively, both of which are

located between the normal C−C single bond and CC

double bond (Table S2). In addition, the dihedral angle

between the electron donor indole quinary ring and the

electron acceptor terpyridine moiety of ZnL1 reveals that it is

of ZnL1 was grown from methanol/chloroform solution. H NMR

(

400 MHz, d -DMSO, ppm): δ 9.19 (s, 4H), 9.06 (d, J = 8.0 Hz, 4H),

.85 (s, 2H), 8.52 (d, J = 4.6 Hz, 2H), 8.29 (t, J = 7.7 Hz, 4H), 7.97

(

d, J = 4.8 Hz, 4H), 7.80 (d, J = 5.2 Hz, 2H), 7.57−7.47 (m, 4H),

.47−7.33 (m, 4H), 4.43 (t, J = 6.7 Hz, 4H), 2.17−1.74 (m, 4H),

.63−1.38 (m, 4H), 0.98 (t, J = 7.3 Hz, 6H). C NMR (100 MHz,

d -DMSO): δ 155.13, 149.82, 149.24, 148.36, 148.22, 148.00, 142.17,

41.82, 141.66, 137.34, 133.07, 130.59, 128.94, 128.72, 128.13,

27.90, 125.13, 123.75, 122.61, 120.72, 119.89, 113.34, 110.92, 45.83,

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

almost a planar structure. In this regard, the resultant enhanced

electron delocalization and charge transfer within ZnL1 will be

conducive to its intensive two-photon activity.

Photophysical Properties. Time-dependent density func-

tional theory (TD-DFT) calculations are implemented to

study the singlet−triplet energy gap (ΔE ) of L1 and ZnL1

Figure S11 ). Evidently, the ΔE values of L1 and ZnL1 are

.08 and 0.83 eV (Figure 1c), respectively. The smaller ΔE_S_T

of ZnL1 suggests its enhanced intersystem crossing (ISC)

process favors the type II process, which gives the eﬃcient O

generation.

9−22

Because the reduction of oxygen to super-

oxide would require the oxidation capacity of the zinc complex,

the redox behavior of organic ligand L1 and the corresponding

complex ZnL1 has been investigated. As shown in Figure S12,

compared with that of L1, the lower oxidation potential of

ZnL1 (1.186 and 0.294 V for L1 and ZnL1, respectively)

makes the superoxide radical production (type I process)

easier.

Figure 2. (a) Two-photon (TP) action cross section of L1 and ZnL1

The UV −vis absorption spectra of L1 and ZnL1 are shown

in Figure S13 . By adjusting the electronic properties of the

coordination metal, an absorption band located at 410 nm

emerged (in DMSO) for ZnL1 compared with that of L1,

which belonged to the ligand-to-metal charge transfer

(

excitation wavelength = 680−900 nm, identical energy = 500 mW).

b) ESR signals of L1/ZnL1 trapped by TEMP with and without light

irradiation (c = 10 μM). (c) ESR signals of ZnL1 trapped by DMPO

with and without light irradiation. (d) The increasing ﬂuorescence

intensity of DHR123 (526 nm) in L1 and ZnL1 solution (10 μM)

(

LMCT) process. Also, the more abundant electron ﬂow

−5

under light irradiation within 6 min (c = 1 × 10 mol/L, λ_e_x= 410

within ZnL1 is beneﬁcial to the type I process. The

ﬂuorescence behaviors of the L1/ZnL1 were measured in

diﬀerent solvents (Figure S13 and Table S3 ). Upon the

excitation at the maximum absorption wavelength, they exhibit

similar emission wavelengths. Interestingly, the ﬂuorescence

quantum yield of ZnL1 was signiﬁcantly lower than that in L1

in diﬀerent solvents which may be due to the nature of

competition between the ISC (S −T ) and ﬂuorescence decay

nm, EX slit = 5.0 nm, EM slit = 5.0 nm).

distinguish the photogenerated ROS species. Accordingly,

2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-

pyrroline-N-oxide (DMPO) were added into the targeted

·−

systems (L1 and ZnL1) to trap as the O and O trapping

agents, respectively. As illustrated in Figure 2b, characteristic

signals of 4-oxo-TEMPO (g = 2.0055, 1:1:1 triplet) under light

(

S −S ) (Figure S14 ). For ZnL1, given its smaller ΔE ,

most of the S excitons convert into the T state favoring the

irradiation for L1 and ZnL1 were displayed, manifesting the

production of O . In addition, the stronger signals of ZnL1

ROS generation instead of ﬂuorescent emission. The stability

of ZnL1 in PBS buﬀers was also checked (Figures S15 and

S16 ). Obviously, as shown in Figure S18a, ZnL1 showed long-

lived excited states with respect to those of L1 (4.25 and 7.24

ns for L1 and ZnL1, respectively), which is conducive to the

reveal its enhanced ISC behavior as a result of the smaller

ΔE . As shown in Figure 2c, no obviously ESR signals could

29−31

be observed for ZnL1 in the dark.

In addition, in contrast

to the negligible signals of the L1 system (Figure S19a),

accordant signals with DMPO−OOH as a spin derivative of

·−

ISC process favoring O₂generation (activated type II

DMPO−O were obtained for ZnL1, suggesting it has the

4,25

·−

process).

ability to reduce O to O₂species. The above results

We, herein, demonstrated that increased electron delocaliza-

tion and charge transfer within ZnL1 would strengthen its two-

photon absorption activity (Figure S17). Frankly, the results

unveiled that the two-photon absorption cross section of ZnL1

was enhanced 1.21 times that of L1 in the wavelength range

from 680 to 900 nm (Figure 2a).

elaborated that, thanks to the coordinated Zn atoms, ZnL1 can

eﬀectively produce singlet oxygen and superoxide radicals.

Moreover, as shown in Figures S19b,c and S20 and Figure 2d,

the generated ROS were further conﬁrmed by the probe 9,10-

anthracenedipropanoic acid (ABDA) and the O₂^·probe

−

dihydrorhodamine 123 (DHR123). The production eﬃcien-

·−

Light Triggered O and O Generation. The above

cies of O by L1/ZnL1 were determined using ABDA as the

results motivated us to study the ROS generation ability of L1

and ZnL1. Therefore, 2′,7′-dichlorodihydroﬂuorescein diac-

O indicator. As we expected, their relative O quantum

yields (Φps) showed diﬀerent values (0.09 and 0.72 for L1 and

etate (H DCF-DA), as an ROS indicator with a lighting-on

ZnL1, respectively) when using Rose Bengal (RB) (Φ = 0.75

signal (green-emitting centered at 525 nm) when reacting with

ROS, was used to detect the ROS generation level of L1 and

in H O) as the standard under cell-free conditions

(respectively, see Figures S20 and S21).

ZnL1. After H DCF-DA was mixed with L1/ZnL1, upon

Intracellular ROS Detection. Beneﬁting from the high-

performance of ZnL1 in producing ROS in vitro, the study to

evaluate the ROS generation of ZnL1 inside HeLa cells was

illumination, the green-emitting signal of H DCF-DA

increased signiﬁcantly, which showed that L1/ZnL1 could

26−28

6,32

produce eﬀective reactive oxygen species.

At the same

carried out next. HeLa cells were incubated with both ZnL1

time, after 10 min of irradiation, it was found that the

ﬂuorescence intensity of ZnL1 was 2.17 times higher than that

of L1 (Figure S18c,d ). This indicated that the capacity of

ZnL1 to generate reactive oxygen species was signiﬁcantly

higher than that of L1. Furthermore, electron spin resonance

and 2′,7′-dichloroﬂuorescein diacetate (DCFH-DA); herein,

DCFH-DA was used as an intracellular ROS indicator. Then,

the resulting cells were exposed to 830 nm of light irradiation

(100 mW/cm ), and then, the confocal laser scanning

microscopy (CLSM) images were collected during incubation

(

ESR) trapping measurements were carried out to further

time. As shown in Figure 3a, the green ﬂuorescent signals of

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

process to produce the longer-lived triplet states ( ZnL1 ). In

Article

Figure 3. (a) CLSM images of HeLa cells treated with DCFH-DA (λ_e_x=₂488 nm, λ = 510−540 nm) followed by the incubation of ZnL1 (10

μM), respectively (two-photon light irradiation = 830 nm, 100 mW/cm ). Scale bar: 20 μm. (b) ROS detection in HeLa cells using DHE and

−

·−

SOSG as the O₂and O probes, respectively. NaN and SOD as the speciﬁc scavengers for O and O , respectively (the boxes in the bright

ﬁeld represent the morphology of cells under diﬀerent conditions). Scale bar: 20 μm.

DCFH-DA demonstrated that cells treated with ZnL1

exhibited eﬀective ROS generation upon light irradiation.

Moreover, the ROS were also studied using standard

dihydroethidium (DHE) and singlet oxygen sensor green

the type I process, ³ZnL1 can react with the biological

environment through the electron transfer mechanism to

produce O₂^·radicals. Meanwhile, for the type II process, the

−

energy of the excited ZnL1 is transferred to molecular oxygen

23,33

(

SOSG) staining methods.

As shown in Figure 3b, after

(O ) to generate singlet oxygen ( O ). These species are highly

exposure to the 830 nm laser light, the bright red ﬂuorescence

of DHE and the green ﬂuorescence of SOSG were observed in

reactive and can trigger cell death resulting from the direct

reaction with the biological surroundings.

34−36

HeLa cells by CLSM. In addition, NaN and superoxide

Cellular Localization. Considering the excellent 2PA and

eﬃcient ROS activities of ZnL1, the biological imaging

application of ZnL1 was carried out using HeLa cells. First,

HeLa cells were incubated with ZnL1 (10 μM) for 15 min. It

can be found that ZnL1 penetrated the cell membrane and

distinct mitochondrial location well (Figure 5a and Figure

dismutase (SOD), applied as the speciﬁc scavengers for O

−

and O₂, were added into the above system. Obviously, NaN₃

and SOD dramatically decreased DHE/SOSG oxidation in the

ZnL1 system. These results demonstrated that the Zn complex

can generate singlet oxygen and superoxide radicals by

photoactivation.

S24a ). To further test the mitochondria-targeting property of

ZnL1, the colocalization imaging experiment using ZnL1 and

MitoTracker Red (a red ﬂuorescent dye that targets

mitochondria) to colabel HeLa cells was performed. As

illustrated in Figure 5b, the distribution of ZnL1 in cells was

consistent with that of MitoTracker Red (Pearson’s colocaliza-

ROS Production Mechanisms. In view of the above

results, we proposed that the pathway of ROS production for

ZnL1 under two-photon excitation is as follows (Figure 4):

During this process, a photosensitizer (ZnL1) absorbs two

near-infrared photons, is excited to the singlet states ( ZnL1)

from its ground states (S ), and then undergoes the ISC

tion coeﬃcient value is 0.95), which demonstrated that

ZnL1 can speciﬁcally localize to mitochondria in HeLa cells.

As reported, the mitochondria-targeting ability of ZnL1 can be

ascribed to its moderate hydrophobicity (log P = 0.26) and

38,39

cationic feature (Figure S22 ).

In Vitro Anticancer Activities and PDT Activities.

Encouraged by the eﬃcient ROS generation and desirable two-

photon activity of ZnL1, we conducted a preliminary test on its

ability to induce apoptosis. First, an MTT method was carried

out to evaluate the PDT eﬃcacy of ZnL1 in vitro. As shown in

Figure S23 , L1 and ZnL1 displayed negligible dark cytotoxicity

(

over 85% of the cells are alive at a concentration of 10 μM).

For photocytotoxicity measurement, HeLa cells were treated

with L1/ZnL1 (10 μM) and exposed to the light. Interestingly,

in contrast that of L1, only about 20% of treated cells survived

for ZnL1. This indicated that ZnL1 exhibited comparable

PDT eﬃcacy in vitro. The PDT eﬀect of ZnL1 was also

Figure 4. Schematic illustration of O generation through the energy

−

transfer process and O₂generation through the electron transfer

process.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

with the ﬂuorescence intensity of the line in the merged image and colocalization coeﬃcient. Scale bar: 20 μ m.

Article

Figure 5. (a) CLSM images of HeLa cells treated with ZnL1 (10 μM) in aqueous phosphate buﬀered saline (PBS) buﬀer at diﬀerent times. (b)

Determination of intercellular localization of ZnL1 by confocal microscopy in HeLa cells. Colocalization images of HeLa cells stained with

MitoTracker Red (0.5 μM, red channel, λ = 534 nm, λ = 600 ± 20 nm) and ZnL1 (10 μM, green channel, λ = 410 nm, λ = 480 ± 20 nm),

Figure 6. (a) Flow cytometry quantiﬁcation of Annexin V-FITC and propidium iodide (PI) labeled HeLa cells. The cells were treated with ZnL1

10 μM) for 12 h before being irradiated. Light group: Cells were processed with light irradiation after treatment with ZnL1 for diﬀerent amounts

(

of time (830 nm, 100 mW/cm ). (b) PDT eﬃcacy of ZnL1 (10 μM) in vitro using Annexin V-FITC/PI (5 μM) treatment (without light

irradiation). HeLa cells in vitro after diﬀerent treatments. ZnL1 incubated with Annexin V-FITC and under PI (830 nm, 100 mW/cm , 15 min).

Scale bar: 20 μm. (c) CLSM images of HeLa cells treated by Lyso-tracker Green (1 μM, λ_e_x= 488 nm, λ_e_m= 490−520 nm) and ZnL1 (10 μM)

with diﬀerent irradiation times (1−6 min). Scale bar: 10 μm.

researched using the ﬂow cytometry cell apoptosis assay to

apoptosis; these results showed high apoptosis enhancement

under light by ZnL1. In addition, we also performed the

Annexin V-FITC/PI experiment to study the PDT eﬃciency.

As shown in Figure 6b, after HeLa cells were incubated with 10

,33

detect cell apoptosis before and after irradiation.

As shown

in Figure 6a, compared with the dark condition, upon light

irradiation at diﬀerent times, ZnL1 resulted in 70.4%

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

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Article

μM ZnL1 for 15 min and then stained with Annexin V-FITC/

PI, ignorable apoptosis signals could be observed without

irradiation. On the contrary, in the PDT group, the red and

green ﬂuorescence intensity signals were detected which

indicated that ZnL1 could induce cancer cell apoptosis

under 830 nm of light irradiation. Considering the positively

charged character of ZnL1, its mitochondria-targeting ability

will play a vital role in cell apoptosis due to the fact that

mitochondria are the main place where cells produce energy,

Accession Codes

CCDC 1919752 and 1959885 contain the supplementary

crystallographic data for this paper. These data can be obtained

free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by

emailing data_request@ccdc.cam.ac.uk, or by contacting The

Cambridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

AUTHOR INFORMATION

Corresponding Authors

■

40,41

and mitochondrial damage can easily lead to apoptosis.

Then, we further studied the behavior of ZnL1 in living cells to

detect the process of cell damage in real time under 830 nm

excitation. It was found that ZnL1 could target mitochondria

well, and the time-resolved imaging with lysosomes showed

that the cell morphology gradually shrank; the ﬁlamentous

mitochondria gradually became punctate after 120 s, which

meant that mitochondria began autophagy (Figure 6c). About

Dandan Li − Institutes of Physics Science and Information

Technology, Key Laboratory of Structure and Functional

Regulation of Hybrid Materials, Ministry of Education, Anhui

University, Hefei 230601, P. R. China; orcid.org/0000-

002-3504-8739 ; Email: chemlidd@163.com

Yupeng Tian − Department of Chemistry, Key Laboratory of

Functional Inorganic Material Chemistry of Anhui Province,

Anhui University, Hefei 230601, P. R. China; Email: yptian@

ahu.edu.cn

60 s later, mitochondria and lysosomes began autophagy.

Lysosomes fuse continuously and eventually form apoptotic

vesicles, leading to apoptosis (Figure S24c).

33,42

According to

the above results, the ZnL1 in our work exhibits good

photostability, multiple ROS types, high singlet oxygen

quantum yields, low dark toxicity, and moderate excitation

wavelengths (Table S5 ). It demonstrated that ZnL1 could be

employed as an eﬃcient two-photon excited ROS generator for

potentially photodynamic therapy.

Authors

Bo Ni − Institutes of Physics Science and Information Technology,

Key Laboratory of Structure and Functional Regulation of

Hybrid Materials, Ministry of Education and Department of

Chemistry, Key Laboratory of Functional Inorganic Material

Chemistry of Anhui Province, Anhui University, Hefei 230601,

P. R. China

CONCLUSIONS

Hongzhi Cao − School of Life Science, Anhui University, Hefei

■

30601, P. R. China

In summary, we have rationally designed and fabricated a

noble-metal-free bis(terpyridine)zinc(II) complex (ZnL1)

bearing two-photon activity. Thanks to the coordination of

the Zn atom, the involved electron transfer (type I) and energy

transfer (type II) processes for ROS generation have been

activated. Moreover, based on the characteristics of cationic

and lipophilic compounds, ZnL1 has outstanding mitochon-

dria-targeting ability. Flow cytometry assay showed that ZnL1

could signiﬁcantly enhance the anticancer activity under two-

photon light irradiation. These results clarify that ZnL1 can

produce ROS upon light irradiation and then cause the cell

death. Overall, this work provides a new platform for switching

on two-photon excited ROS generation without the

introduction of heavy atoms.

Chengkai Zhang − Department of Chemistry, Key Laboratory of

Functional Inorganic Material Chemistry of Anhui Province,

Anhui University, Hefei 230601, P. R. China

Shengli Li − Department of Chemistry, Key Laboratory of

Functional Inorganic Material Chemistry of Anhui Province,

Anhui University, Hefei 230601, P. R. China

Qiong Zhang − Department of Chemistry, Key Laboratory of

Functional Inorganic Material Chemistry of Anhui Province,

Anhui University, Hefei 230601, P. R. China

Xiaohe Tian − School of Life Science, Anhui University, Hefei

30601, P. R. China; orcid.org/0000-0002-2294-3945

Jieying Wu − Department of Chemistry, Key Laboratory of

Functional Inorganic Material Chemistry of Anhui Province,

Anhui University, Hefei 230601, P. R. China

https://pubs.acs.org/10.1021/acs.inorgchem.0c02030

ASSOCIATED CONTENT

■

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c02030.

Author Contributions

B.N and H.Z.C. contributed equally to this work.

Notes

General experimental details, synthetic procedures,

characterization data, crystal structure, optical physical

data, ROS detection, HNMR sprectra, CNMR spectra,

mass spectra, ORTEP structures, HOMO and LUMO

distributions, cyclic voltammetry (CV), UV−vis absorp-

tion spectra, quantum yield, time evolution of UV−vis

absorption spectra, stability of complexes, two-photon

ﬂuorescence intensity, ﬂuorescence lifetime spectra, ESR

signals, chemical trapping measurements, decomposition

rate constants, octanol/water partition coeﬃcient, HeLa

cell toxicity data, single photon confocal development,

crystal data and structure reﬁnement, selected bond

lengths, photophysical data, dark toxicity and photo-

toxicity, and coordinates of the atoms (PDF)

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by grants for the National Natural

Science Foundation of China (21871003, 21701160,

1672002, and 51772002), Doctor Start-up Fund

(

S020118002/026), Hefei National Laboratory for Physical

Sciences at the Microscale (KF2019002), and Natural Science

Foundation of Anhui Province of China (1908085MB30).

REFERENCES

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1) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic

therapy for cancer. Nat. Rev. Cancer 2003, 3, 380−387.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

2) Bonnett, R. Photosensitizers of the porphyrin and phthalocya-

nine series for photodynamic therapy. Chem. Soc. Rev. 1995, 24, 19−

3) Lan, M.; Zhao, S.; Liu, W.; Lee, C. S.; Zhang, W.; Wang, P.

Photosensitizers for photodynamic therapy. Adv. Healthcare Mater.

019, 8, 1900132.

4) Awuah, S. G.; You, Y. Boron dipyrromethene (BODIPY)-based

photosensitizers for photodynamic therapy. RSC Adv. 2012, 2,

1169−11183.

5) Dai, Y.; Yang, Z.; Cheng, S.; Wang, Z.; Zhang, R.; Zhu, G.;

(19) Li, C.; Ren, Z.; Yan, S. Thermally activated delayed

fluorescence materials in OLEDs devices: Design, synthesis and

applications. Kexue Tongbao 2015, 60, 2989−3004.

(20) Zhang, C.; Zhao, Y.; Li, D.; Liu, J.; Han, H.; He, D.; Tian, X.;

Li, S.; Wu, J.; Tian, Y. Aggregation-induced emission (AIE)-active

molecules bearing singlet oxygen generation activities: the tunable

singlet-triplet energy gap matters. Chem. Commun. 2019, 55, 1450−

1453.

Z.; Li, H.; Deng, R.; Liu, X.; Huang, W. Stabilizing triplet excited

(21) An, Z.; Zheng, C.; Tao, Y.; Chen, R.; Shi, H.; Chen, T.; Wang,

states for ultralong organic phosphorescence. Nat. Mater. 2015, 14,

685−690.

Wang, Z.; Yung, B. C.; Tian, R.; Jacobson, O.; et al. Toxic Reactive

Oxygen Species Enhanced Synergistic Combination Therapy by Self-

Assembled Metal-Phenolic Network Nanoparticles. Adv. Mater. 2018,

(22) Li, J.-A.; Zhou, J.; Mao, Z.; Xie, Z.; Yang, Z.; Xu, B.; Liu, C.;

Chen, X.; Ren, D.; Pan, H.; Shi, G.; Zhang, Y.; Chi, Z. Transient and

Persistent Room-Temperature Mechanoluminescence from a White-

Light-Emitting AIEgen with Tricolor Emission Switching Triggered

by Light. Angew. Chem., Int. Ed. 2018, 57, 6449−6453.

(

0, 1704877.

6) Monro, S.; Colon, K. L.; Yin, H.; Roque, J., III; Konda, P.; Gujar,

S.; Thummel, R. P.; Lilge, L.; Cameron, C. G.; McFarland, S. A.

Transition metal complexes and photodynamic therapy from a tumor-

centered approach: Challenges, opportunities, and highlights from the

development of TLD1433. Chem. Rev. 2019 , 119 , 797 −828.

(23) Li, M.; Xia, J.; Tian, R.; Wang, J.; Fan, J.; Du, J.; Long, S.; Song,

X.; Foley, J. W.; Peng, X. Near-infrared light-initiated molecular

superoxide radical generator: rejuvenating photodynamic therapy

against hypoxic tumors. J. Am. Chem. Soc. 2018, 140, 14851−14859.

8) Sun, Z.; Zhang, L. P.; Wu, F.; Zhao, Y. Photosensitizers for Two-

7) DeRosa, M. C.; Crutchley, R. Photosensitized singlet oxygen and

its applications. Coord. Chem. Rev. 2002, 233 , 351 −371.

Photon Excited Photodynamic Therapy. Adv. Funct. Mater. 2017, 27,

704079.

9) Kim, H. M.; Jung, C.; Kim, B. R.; Jung, S. Y.; Hong, J. H.; Ko, Y.

(24) Mao, Z.; Yang, Z.; Fan, Z.; Ubba, E.; Li, W.; Li, Y.; Zhao, J.;

Yang, Z.; Aldred, M. P.; Chi, Z. The methylation effect in prolonging

the pure organic room temperature phosphorescence lifetime. Chem.

Sci. 2019, 10, 179−184.

(

(25) Yang, Z.; Mao, Z.; Zhang, X.; Ou, D.; Mu, Y.; Zhang, Y.; Zhao,

G.; Lee, K. J.; Cho, B. R. Environment-sensitive two-photon probe for

intracellular free magnesium ions in live tissue. Angew. Chem., Int. Ed.

(

Cho, B. R. Ratiometric detection of mitochondrial thiols with a two-

photon fluorescent probe. J. Am. Chem. Soc. 2011, 133, 11132−11135.

Modification of side chain of conjugated molecule for enhanced

charge transfer and two-photon activity. Spectrochim. Acta, Part A

(

Ji, L.; Chao, H. Highly Charged Ruthenium (II) Polypyridyl

Complexes as Lysosome-Localized Photosensitizers for Two-Photon

Photodynamic Therapy. Angew. Chem., Int. Ed. 2015, 54, 14049−

action of Ru (II) polypyridyl complexes in living cells upon light

irradiation. Chem. Commun. 2018, 54, 13040−13059.

Markova, L.; et al. Towards Novel Photodynamic Anticancer Agents

photocleavage by an osmium (II) complex in the PDT window. Chem.

Commun. 2010, 46, 6759−6761.

(

G.; Gandioso, A.; Svitelova, M.; Bresolí-Obach, R.; Kostrhunova, H.;

Generating Superoxide Anion Radicals: A Cyclometalated Ir

C.; Liu, S.; Chi, Z.; Xu, J.; Wu, Y.-C.; Lu, P.-Y.; Lien, A.; Bryce, M. R.

Intermolecular Electronic Coupling of Organic Units for Efficient

Persistent Room-Temperature Phosphorescence. Angew. Chem., Int.

Ed. 2016, 55, 2181−2185.

007, 46, 3460−3463.

10) Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.;

26) Apel, K.; Hirt, H. Reactive oxygen species: metabolism,

oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004,

5, 373−399.

27) Andreyev, A.; Kushnareva, Y.; Starkov, A. Mitochondrial

metabolism of reactive oxygen species. Biochemistry (Moscow) 2005,

0, 200−214.

28) Zheng, Z.; Zhang, T.; Liu, H.; Chen, Y.; Kwok, R. T. K.; Ma,

11) Li, D.; Li, B.; Wang, S.; Zhang, C.; Cao, H.; Tian, X.; Tian, Y.

S.; Tang, B. Z. Bright near-infrared aggregation-induced emission

020, 224, 117448.

12) Huang, H.; Yu, B.; Zhang, P.; Huang, J.; Chen, Y.; Gasser, G.;

(

C.; Zhang, P.; Sung, H. H. Y.; Williams, I. D.; Lam, J. W. Y.; Wong, K.

luminogens with strong two-photon absorption, excellent organelle

specificity, and efficient photodynamic therapy potential. ACS Nano

4052.

13) Jakubaszek, M.; Goud, B.; Ferrari, S.; Gasser, G. Mechanisms of

(

018, 12, 8145−8159.

29) Li, M.; Xia, J.; Tian, R.; Wang, J.; Fan, J.; Du, J.; Long, S.; Song,

X.; Foley, J. W.; Peng, X. Near-infrared light-initiated molecular

superoxide radical generator: rejuvenating photodynamic therapy

against hypoxic tumors. J. Am. Chem. Soc. 2018 , 140 , 14851 −14859.

14) Sun, Y.; Joyce, L. E.; Dickson, N. M.; Turro, C. DNA

(30) Buettner, G. The Spin Trapping of Superoxide and Hydroxyl

15) Novohradsky, V.; Rovira, A.; Hally, C.; Galindo, A.; Vigueras,

Free Radicals with DMPO (5,5-Dimethylpyrroline-N-oxide): More

About Iron. Free Radical Res. Commun. 1993, 19, No. s79-s87.

(31) Chen, Y.; Wang, Z.; Wang, H.; Lu, J.; Yu, S.; Jiang, H. Singlet

III

Oxygen-Engaged Selective Photo-Oxidation over Pt Nanocrystals/

Porphyrinic MOF: The Roles of Photothermal Effect and Pt

Electronic State. J. Am. Chem. Soc. 2017, 139, 2035−2044.

Complex Conjugated to a Far-Red Emitting Coumarin. Angew.

Chem., Int. Ed. 2019, 58, 6311−6315.

(

16) Xiao, Y.-F.; Chen, J.-X.; Li, S.; Tao, W.-W.; Tian, S.; Wang, K.;

(32) Li, X.; Wu, J.; Wang, L.; He, C.; Chen, L.; Jiao, Y.; Duan, C.

Cui, X.; Huang, Z.; Zhang, X.-H.; Lee, C.-S. Manipulating exciton

dynamics of thermally activated delayed fluorescence materials for

tuning two-photon nanotheranostics. Chem. Sci. 2020, 11, 888−895.

Mitochondrial-DNA-Targeted Ir(III)-Containing Metallohelices with

Tunable Photodynamic Therapy Efficacy in Cancer Cells. Angew.

Chem. 2020, 132 (16), 6482−6489.

(

17) Starkey, J.; Rebane, A.; Drobizhev, M.; Meng, F.; Gong, A.;

(33) Novohradsky, V.; Vigueras, G.; Pracharova, J.; Cutillas, N.;

Elliott, A.; McInnerney, K.; Spangler, C. New two-photon activated

photodynamic therapy sensitizers induce xenograft tumor regressions

after near-IR laser treatment through the body of the host mouse.

Clin. Cancer Res. 2008, 14, 6564−6573.

Janiak, C.; Kostrhunova, H.; Brabec, V.; Ruiz, J.; Kasparkova, J.

Molecular superoxide radical photogeneration in cancer cells by

dipyridophenazine iridium (iii) complexes. Inorg. Chem. Front. 2019,

6, 2500−2513.

(

18) Chen, R.; Zhang, J.; Chelora, J.; Xiong, Y.; Kershaw, S. V.; Li,

(34) Liu, J.; Bu, W.; Shi, J. Chemical design and synthesis of

functionalized probes for imaging and treating tumor hypoxia. Chem.

Rev. 2017, 117, 6160−6224.

K. F.; Lo, P.-K.; Cheah, K. W.; Rogach, A. L.; Zapien, J. A.; Lee, C.-S.

Ruthenium (II) Complex Incorporated UiO-67 Metal −Organic

Framework Nanoparticles for Enhanced Two-Photon Fluorescence

Imaging and Photodynamic Cancer Therapy. ACS Appl. Mater.

Interfaces 2017, 9, 5699−5708.

(35) Zhou, Z.; Song, J.; Nie, L.; Chen, X. Reactive oxygen species

generating systems meeting challenges of photodynamic cancer

therapy. Chem. Soc. Rev. 2016, 45, 6597−6626.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Liu, S.; Tang, B.; Liu, X.; Su, Z.; Bryce, M. AIE Multinuclear Ir(III)

Article

(

36) Zhang, L.; Li, Y.; Che, W.; Zhu, D.; Li, G.; Xie, Z.; Song, N.;

Complexes for Biocompatible Organic Nanoparticles with Highly

Enhanced Photodynamic Performance. Adv. Sci. 2019 , 6 , 1802050.

37) Huang, H.; Tian, Y. A ratiometric fluorescent probe for

bioimaging and biosensing of HBrO in mitochondria upon oxidative

stress. Chem. Commun. 2018, 54, 12198−12201.

38) Horobin, R. W.; Stockert, J. C.; Rashid-Doubell, F. Fluorescent

cationic probes for nuclei of living cells: why are they selective? A

quantitative structure −activity relations analysis. Histochem. Cell Biol.

(

006, 126, 165−175.

39) Feng, Z.; Li, D.; Zhang, M.; Shao, T.; Shen, Y.; Tian, X.; Zhang,

Q.; Li, S.; Wu, J.; Tian, Y. Enhanced three-photon activity triggered

by the AIE behaviour of a novel terpyridine-based Zn(II) complex

bearing a thiophene bridge. Chem. Sci. 2019, 10 (30), 7228 −7232.

40) Oleinick, N.; Morris, R.; Belichenko, I. The role of apoptosis in

response to photodynamic therapy: what, where, why, and how.

Photochem. Photobiol. Sci. 2002, 1, 1−21.

41) Li, Y.; Wu, X.; Liu, X.; Li, P. Mitophagy imbalance in

cardiomyocyte ischaemia/reperfusion injury. Acta Physiol. 2019, 225,

No. e13228.

(

42) Jung, H. S.; Han, J.; Lee, J.-H.; Lee, J. H.; Choi, J.-M.; Kweon,

H.-S.; Han, J. H.; Kim, J.-H.; Byun, K. M.; Jung, J. H.; Kang, C.; Kim,

J. S. Enhanced NIR radiation-triggered hyperthermia by mitochon-

drial targeting. J. Am. Chem. Soc. 2015, 137, 3017−3023.