moPlatinum-AIEgen Coordination Complex for Imaging-Guided Annihilation of Cisplatin-Resistant Cancer Cells

Zhenyan He‡a, Yuting Gao‡a, Haoke Zhangb, Xiuxia Wanga, Fanling Menga, Liang Luo*a, and Ben Zhong Tang*b

a. National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong

University of Science and Technology, Wuhan, 430074, China. E-mail: liangluo@hust.edu.cn

b. Department of Chemistry and Hong Kong Branch of Chinese National Engineering Research Center for

Tissue Restoration and Reconstruction, The Hong Kong University of Science & Technology, Clear Water Bay,

Kowloon, Hong Kong, China. E-mail: tangbenz@ust.hk Kong, China.E-mail: tangbenz@ust.hk‡ These authors contributed equally to this work

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2020

Contents:1. Experimental Section .............................................................................................S-1

1.1 Materials and Instruments...................................................................................S-1

1.2 Experimental Methods ...................................................................................... S-2

2.Schemes and Figures...............................................................................................S-6

Scheme S1................................................................................................................S-6

Fig. S1. .................................................................................................................S-7

Fig. S2. .................................................................................................................S-7

Fig. S3. .................................................................................................................S-8

Fig. S4. .................................................................................................................S-8

Fig. S5. .................................................................................................................S-8

Fig. S6. .................................................................................................................S-9

Fig. S7. ................................................................................................................ S-9

Fig. S8. .................................................................................................................S-10

Fig. S9. ................................................................................................................ S-11

Fig. S10. ............................................................................................................ S-11

Fig. S11. .............................................................................................................S-12

Fig. S12. .............................................................................................................S-12

Fig. S13. .............................................................................................................S-13

Fig. S14. .............................................................................................................S-13

Fig. S15. .............................................................................................................S-14

Fig. S16. .............................................................................................................S-14

Table S1 ...........................................................................................................S-14

Fig. S17. .............................................................................................................S-15

Fig. S18. .............................................................................................................S-15

Table S2 .............................................................................................................S-16

References ..............................................................................................................S-16

S-1

Experimental Section

1. Materials and Instruments

Materials: All organic reagents were purchased from Energy Chemical (China). All

solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. Unless noted all

reagents and solvents with analytical purity were used as received. Bovine serum

albumin (BSA) was purchased from Biosharp. Lysozyme was purchased from Sigma-

Aldrich. 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl tetrazolium bromide (MTT) was

purchased from Boster Biological Teachnology Co., Ltd. Lysotracker Red was

obtained from Yeasen Biotechnology Co., Ltd. (Shanghai). A549 and A549/DDP

cells were purchased from Shanghai Mito Biological Teachnology Co., Ltd.

HeLa/DDP cells was purchased from Shcqsw biomart Co., Ltd. Nuclear Extraction

Kit was purchased from Beijing Solarbio Science and Technology Co.,Ltd.

Instruments: All nuclear magnetic resonance (NMR) spectra were recorded on an

Agilent 400-MR 400 MHz spectrometer operated in the Fourier transform mode.

DMSO-d6 was used as the solvent. High-resolution electrospray mass spectroscpy

(HR-ESI-MS) was conducted on a SolariX 7.0T mass spectrometer (BrukerDaltonics,

USA). UV-Vis absorption spectra were acquired on a TU-1810DSPC UV/Vis

spectrophotometer (Puxi General Instrumental Company, China). TEM images were

recorded on transmission electron microscope (Hitachi HT7700, Japan). Fluorescence

spectra were recorded on a HITACHI F4600 fluorescence spectrophotometer.

Cytotoxicity data were recorded on a microplate reader (Varioskan LUX, Thermo

S-2

Scientific, USA). Inductively Coupled Plasma Optical Emission Spectrometer (ICP-

OES) was conducted on Agilent 700.

2. Experimental Methods

Synthesis of TPCTP: The intermediate (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(4-(bis (4-

(pyridin-4-yl)phenyl)amino)phenyl)acrylonitrile) (TPC) was synthesized following a

literature procedure.[1] In a 25 mL round-bottom flask, a mixture of cisplatin (10.20

mg, 0.034 mmol) and silver nitrate (5.78 mg, 0.034 mmol) in N,N-

dimethylformamide (DMF) (10 mL) was stirred and heated to 60 °C for 24 h under a

nitrogen atmosphere. After cooling to room temperature, the reaction mixture was

separated by centrifugation. The supernatant was added with TPC (3.32 mg, 0.0034

mmol) and stirred at 60 °C for 24 h under a nitrogen atmosphere. After cooling to

room temperature, the mixture was poured into diethyl ether. The precipitate was then

washed with diethyl ether three times and dried under reduced pressure to afford the

product as an orange powder (6.16 mg, yield: 89%).

The value of water solubility: Frist, the standard curve of TPC and TPCTP in

dimethyl sulfoxide (DMSO) have been determined. Then, TPC and TPCTP (0.5 mg)

were dissolved in pure water (8 mL), respectively. The aqueous solution was gently

swung for 24 h (37 °C) after sonication. Then, 6.8 mL of supernatant was lyophilized

after centrifugation. Lyophilized TPC and TPCTP were re-dissolved in DMSO to

measure the absorption value. Finally, the values of water solubility were calculated

according to the standard curve of TPC and TPCTP, respectively.

Measurement of 1O2 generation: The 1O2 generation of TPC and TPCTP in water

S-3

upon white light irradiation (350-800 nm, 1 mW cm-2) was determined using 9,10-

anthracenediyl-bis(methylene) dimalonic acid (ABDA) as an indicator and Rose

Bengal (RB) as the standard reference. [2] The concentration of ABDA was 100 µM

and the concentration of the individual solution of cisplatin, TPC, TPCTP, and RB

was 5 µM. The absorbance decrease of ABDA at 379 nm was recorded upon different

irradiation time. The 1O2 quantum yield was calculated using the following equation

Ф𝑠𝑎𝑚𝑝𝑙𝑒=Ф𝑅𝐵 ×𝐾𝑠𝑎𝑚𝑝𝑙𝑒 ∗ 𝐴𝑅𝐵𝐴𝑠𝑎𝑚𝑝𝑙𝑒 ∗ 𝐾𝑅𝐵

where K is the slope of the absorbance versus irradiation time. A represents the

integral area of absorption by TPC, cisplatin, RB and TPCTP on white irradiation.

ΦRB is the 1O2 quantum yield of reference Rose Bengal, which is 0.75 in water.

KTPCTP, KTPC and KRB were calculated to be 0.00283, 0.00052 and 0.00135,

respectively (Fig. 12D), and ATPCTP, ATPC and ARB during 350-800 nm were

determined to be 24.602, 26.231 and 11.772, respectively (Fig. S8).

Cellular Imaging: All cisplatin-resistant and nonresistant cells were cultured in

chamber (LAB-TEK, Chambered Cover Glass System) with standard medium

containing 10% FBS and 1% antibiotics (37 °C, 5 % CO2). After 24 h, the medium

was replaced with TPCTP in DMEM (5 μM) and incubated for 24 h. Then, the cells

were then treated with Lysotraker Red (100 × 10 −9 M) and incubated for 30 min.

After being washed by phosphate-buffer saline (PBS), the living cells were observed

by confocal laser scanning microscope (CLSM, Perkin Elmer & Olympus,

UltraVIEW VoX & IX81). The excitation wavelength was 488 nm and signal

collection wavelength region was 500-570 nm for TPCTP. The excitation wavelength

S-4

was 594 nm and signal collection wavelength region was 610-710 nm for Lysotracker

Red.

Flow Cytometry Analysis: All cisplatin-resistant and nonresistant cancer cells were

seeded at a density of 2×105 cells/mL of the DMEM medium with 10% FBS on 12-

well plates to the final volume of 1 mL. The plates were incubated for 24 h and then

treated with TPCTP for different time. After incubation under the condition (5% CO2,

37 °C), cells were collected and washed with PBS for twice. The cell uptake was then

quantified by a flow cytometer (CytoFLEX S), immediately.

ICP-OES: HeLa and A549 cells were seeded in 100 mm diameter Petri dish in

triplicate with 3.0 million cells and were incubated in standard medium for 24 h (37

ºC, 5% CO2). Then, the cells were incubated with TPCTP (4 µM) or standard medium

in the dark for 24 h. The cells were treated with trypsin and washed with PBS, and

their nuclei were isolated using the Nuclear Extraction Kit. The samples were then

lyophilized and weighted. Platinum concentrations in all of the fractions were

determined by ICP-OES.

Dark toxicity of TPCTP in Vitro: To evaluate the dark toxicity of TPCTP, the cell

viability of TPCTP to various cisplatin-resistant and nonresistant cells were

investigated. Cells were seeded in 96-well plates and cultured in standard medium for

24 h (37 ºC, 5% CO2). The cells were treated with various concentrations of

individual drug for 48 h in the dark. After been washed by PBS, 100 µL of freshly

prepared MTT solution was added into each well. The MTT solution was carefully

removed after 4 h of incubation, and DMSO (130 µL) was added into each well to

S-5

dissolve all the formazan formed. The absorbance of MTT at 570 nm was measured

by the microplate reader. Cell viability was expressed by the ratio of the cells

incubated with different drugs to those incubated with culture normal medium. Each

experiment was repeated at least three times.

Phototoxicity of TPCTP in Vitro: The cytotoxicity of different drugs in the presence

of white light irradiation was assessed by MTT assays. All cisplatin-resistant and

nonresistant cancer cells were seeded in 96-well plates and cultured in standard media

for 24 h. The cells were then incubated with drug solutions of various concentrations

in the dark for 24 h. The mixtures were exposed to white light irradiation (350-800

nm, 4 mW cm-2) for 30 min. The cells were further cultured for 24 h after irradiation.

After being washed by PBS, 100 µL of freshly prepared MTT solution was added into

each well. The MTT solution was carefully removed after 4 h of incubation, and

DMSO (130 µL) was added into each well to dissolve all the formazan formed. The

absorbance of MTT at 570 nm was measured by the microplate reader.

The Value of Combination Index (CI): To verify whether TPCTP has a synergistic

effect on PDT and chemotherapy, the CI value of TPCTP was calculated by Calcusyn

software. The phototoxicity of TPCTP was caused by the synergy between PDT (TPC

+ Light group) and chemotherapy (cisplatin group). The calculated CI values are

obtained with fraction affected by the dose (Fa) at 0.5.

S-6

Supplementary Schemes and Figures

N

N CN

NC

N

N

N

N

Pt

NH3

Cl

Cl

NH3

4+

N

N CN

NC

N

N

N

N

Pt

Pt

Pt

Pt

NH3

Cl

H3N

H3N NH3

Cl

H3N NH3

ClNH3H3N

Cl

4 NO3-

4+

AgNO3

DMF

TPC Cisplatin

Scheme S1. Synthesis route to TPCTP.

S-7

Fig. S1 The 1H NMR spectra of cisplatin, TPC, and TPCTP. Solvent: DMSO-d6.

Fig. S2 HR-ESI-MS mass spectrum of TPCTP.

4.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

4.84

7.80

8.60

12.0

24.

092.

09

8.00

7.20

7.22

7.28

7.307.

727.

747.

867.

887.

978.

09

8.63

8.64

4.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

11.7

8

11.6

0

12.5

6

25.7

6

8.00

4.33

4.707.21

7.23

7.26

7.28

7.30

7.327.

917.

937.

967.

98

8.72

4.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

0.47

1.00

3.934.

64

400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

Inte

nsity

(×10

8 )

m/z

508.3381

S-8

400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

Fluo

resc

ence

Abs

wavelength (nm)

TPCTP Abs TPC Abs TPCTP FL TPC FL

Fig. S3 UV-vis absorption and fluorescence spectra of TPC and TPCTP.

Concentration: 5 µM.

1 m 1 m 1 m

A B C

Fig. S4 TEM images of TPCTP with concentrations of 1 M (A), 10 M (B) and 50

M (C), respectively. Scale bar: 1 m.

500 600 700 8000

1

2

3

4

5

6

40 50 60 70 80 90

Fraction of water (%)

Fluo

resc

ence

Wavelength(nm)

0 10 20 30

Fig. S5 Fluorescence spectra of TPC (10 µM) in water/THF mixed solvents with

S-9

different water fractions. λex: 440 nm.

Fig. S6 Fluorescence spectra of TPCTP (1 µM) in water/THF mixed solvents with

different water fractions. λex: 440 nm.

0.0

0.5

1.0

1.5

2.0

850750650550

0 10 20 30 40 50 100 150 200

Fluo

resc

ence

Wavelength (nm)

Lysozyme g/mL

450

Fig. S7 Fluorescence spectra of TPCTP (10 µM) in the presence of different

concentrations (0-200 µg/mL) of lysozyme in aqueous solution. λex = 440 nm.

0.0

0.5

1.0

1.5

2.0

850750650550

60 70 80 90 100

0 10 20 30 40 50

Fluo

resc

ence

Wavelength (nm)

Fraction of water (%)

450

S-10

Fig. S8 Absorption spectra of ABDA (100 μM) in the presence of TPCTP (A), TPC

(B), RB (C), and cisplatin (D) after being exposed to white light irradiation for

different time.

300 400 500 6000.00.20.40.60.81.01.2

0 s

A 379

nm

Wavelength (nm)

TPCTPirradiation time(s)

240 s

300 400 500 6000.0

0.2

0.4

0.6

0.8

1.0

0 s

240 sA 379

nm

Wavelength (nm)

RBirradiation time(s)

300 400 500 6000.00.20.40.60.81.01.2

0 s

A 379

nm

Wavelength (nm)

TPCirradiation time(s)

240 s

300 400 500 6000.0

0.2

0.4

0.6

0.8

1.0

240 s

0 sA 37

9 nm

Wavelength (nm)

cisplatinirradiation time(s)

A B

DC

S-11

LUMO = -2.72154 eV

HOMO = -5.53887 eV

N

N CN

NC

N

N

N

NA

B

Fig. S9 HOMO-LUMO distribution of TPC. (A) Chemical structure of TPC. (B)

Optimized structures of the HOMO and LUMO at S1 were calculated by DFT

(Gaussian 09 B3LYP/6-311G(d,p)). HOMO: highest occupied molecular orbital,

LUMO: lowest unoccupied molecular orbital. The HOMO-LUMO transition matched

well with the absorption for TPC.

LUMO = -11.0788 eV

HOMO = -11.2875 eV

N

N CN

NC

N

N

N

NPt

Cl

NH3H3N

PtCl

H3N NH3

PtCl

H3NNH3

PtCl

NH3H3N

A

B

Fig. S10 HOMO-LUMO distribution of TPCTP. (A) Chemical structure of TPCTP.

(B) Optimized structures of the HOMO and LUMO at S1 were calculated by DFT

(Gaussian 09 B3LYP/6-311G(d,p)). HOMO: highest occupied molecular orbital,

LUMO: lowest unoccupied molecular orbital. The interplay became much more

S-12

complicated when TPC was coordinated with metals to form the complex, and it was

difficult to obtain a reasonable energy and electron density distribution of TPCTP.

MergeLysotrackerTPCTP

Fig. S11 CLSM images of A549 cells incubated with TPCTP and Lysotracker Red for

24 h. (TPCTP: λex = 488 nm, λem = 510-570 nm; Lysotracker Red: λex = 594 nm, λem =

610-710 nm). Scale bar: 20 µm.

Fig.S12 CLSM images of HeLa cells incubated with TPCTP and Lysotracker Red

for 24 h. (TPCTP: λex = 488 nm, λem = 510-570 nm; Lysotracker Red: λex = 594 nm,

λem = 610-710 nm). Scale bar: 20 µm.

TPCTP Lysotracker Merge

S-13

Fig. S13 Flow cytometry analysis of A549 cells (top) and A549/DDP (bottom)

treated with TPCTP (5 μM) for different time.

Fig. S14 Flow cytometry study of HeLa cells (top) and HeLa /DDP (bottom) treated

with TPCTP (5 μM) for different time.

Blank 0.5 h 2 h 4 h 24 h

Blank 0.5 h 2 h 4 h 24 h

S-14

0.01 0.1 1 100

20

40

60

80

100 B

Cisplatin TPC + light TPCTP + light

Cel

l via

bilit

y (%

)

Drug concentration (M)

A

0.01 0.1 1 100

20

40

60

80

100

Cel

l via

bilit

y (%

)

Drug concentration (M)

Cisplatin TPC + light TPCTP + light

Fig.S15 MTT assays of A549 cells (A) and HeLa cells (B) pretreated with a series

doses of cisplatin, TPC and light irradiation, and TPCTP and light irradiation. Light

irradiation: 350-800 nm, 4 mW cm-2, 30 min.

Fig. S16 (A) MTT assays of A549 and A549/DDP cells pretreated with a series of

doses of TPCTP in the dark. (B) MTT assays of HeLa and HeLa/DDP cells pretreated

with a series of doses of TPCTP in dark.

Table S1. IC50 (µM) values of cisplatin and TPCTP without/with irradiation in non-

and cisplatin-resistant cancer cells.

Compound A549 A549/DDP HeLa HeLa/DDP

cisplatin 10.1+1.6 32.8+3.95 3.9+0.13 48.2+2.41

TPCTP >20 19.7 19.9 >>20

TPCTP+L 5.1+0.37 5.6+0.17 1.7+0.09 1.7+0.13

0.01 0.1 1 100

20

40

60

80

100

Drug concentration (μM)

A549 A549/DDPC

ell v

iabi

lity

(%)

0.01 0.1 1 100

20

40

60

80

100

Drug concentration (μM)

HeLa HeLa/DDPC

ell v

iabi

lity

(%)

A B

S-15

A549A549/DDP

HeLaHeLa/DDP0.0

0.2

0.4

0.6

0.8

1.0

Com

bina

tion

Inde

x

Fig. S17 CI values of TPCTP in cisplatin-resistant and nonresistant A549 and HeLa

cells. Light irradiation: 350-800 nm, 4 mW cm-2, 30 min.

A549 HeLa0

5

10

15

20

RF

cisplatin TPCTP

Fig. S18 The RF (resistant factor) values for cisplatin and TPCTP in A549 cells and

HeLa cells. RF is defined by the ratio of the IC50 value in drug-resistant cells over

IC50 value in drug nonresistant cells.

S-16

Table.S2 Quantification of platinum inside cells after treatment for 24h by ICP-OES

measurements

Sample name Net nuclei/mg

[Pt] (ng/mg)

HeLa 1.7 0.8blank

A549 3.5 0.61 0.8 4750.02 1.1 6645.13 0.7 8869.1

HeLanuclei

Average 6754.7+1683.41 1.1 842.42 0.9 1751.53 0.7 1070.5

A549nuclei

Average 1221.4+386.2

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