hypoxia regulation and photodynamic therapy electronic

Electronic Supplementary Information (ESI)

Oxygen self-sufficient NIR-activatable liposomes for tumor

hypoxia regulation and photodynamic therapy

Qi Yu,‡a Tianci Huang,‡a Chao Liu,a Menglong Zhao,a Mingjuan Xie,a Guo Li,a Shujuan Liu,a Wei Huang*a,b and Qiang Zhao*a

a. Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key

Laboratory for Biosensors, Institute of Advanced Materials (IAM), Nanjing University

of Posts and Telecommunications (NUPT), Nanjing 210023, P. R. China.

b. Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical

University (NPU), Xi'an 710072, Shaanxi, P. R. China.

Email: [email protected]; [email protected].

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2019

1. Materials

NH4HCO3, DPPC and cholesterol were purchased from Sigma-Aldrich. DSPE-PEG5000

were purchased from Xi’an Ruixi Biological Technology Co., Ltd. CaCl2 and H2O2 (30

w.t. % in water) were purchased from Aladdin. Cell culture reagents and fetal bovine

serum (FBS) were supplied from Beyotime Biotechnology. All reagents were

purchased from commercial suppliers and used without further purification.

2. Instruments

NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz NMR instrument

(1H: 400 MHz, 13C:100 MHz). Chemical shifts (ppm) were reported relative to

tetramethylsilane (TMS). Mass spectra were obtained on a Bruker autoflex matrix-

assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOFMS).

TEM images were obtained from Hitachi TEM system. The average hydrodynamic size

was recorded on ZetaPALS analyser (Brookhaven, USA). XRD analysis were performed

via a Bruker D8 Advanced. UV-Vis absorption and photoluminescence spectra were

measured via a Shimadzu UV-3600 UV-VIS-NIR spectrophotometer and Edinburgh FL

920 spectrophotometer, respectively. The photothermal effects were recorded using

a thermal infrared imager (FLIR E40). The concentration of oxygen generated in

aqueous solution were quantified by a dissolved oxygen meter (HY30D53301000,

HACH, USA). Confocal luminescence images carried out by a laser scanning confocal

microscopy (Olympus Fluo view FV1000) equipped with 20⨯ objective lens.

Photographs of the mice were taken with a Cannon EOC 400D digital camera.

3. Synthesis and characterization of B1

CH3NO2, Et2NHCH3OH, 7 h

NH

O

O

N

N

O

O

CHO

OC6H13

COCH3

OC6H13

+CH3CH2OH, NaOH

30 oC, 9 h

C6H13

C6H13 C6H13

C6H13

N

O

O

N

N

O

OC6H13

C6H13 C6H13

C6H13

BF3.OEt2, DCM, Et3N

rt, 24 hB

N

O

O

N

N

O

OC6H13

C6H13 C6H13

C6H13

BI I

CHCl3, CH3COOH

NIS

1 2

3 4 B1

CH3COONH4, CH3(CH2)3OH80 oC, 12 h

F F F F

C6H13O

O

OC6H13

C6H13O

O

OC6H13

NO2

Compound 1-4 and B1 were synthesized according to the previous work.1

Compound 1: 1H NMR (400MHz, CDCl3) δ (ppm) : 8.02 (d, J = 7.2 Hz, 2 H), 7.77 (d, J =

15.6 Hz, 1 H), 7.58 (d, J = 8.4 Hz, 2 H), 7.43 (d, J = 15.6 Hz, 1 H), 6.96 6.91 (m, 4 H),

4.08 3.97 (m, 4 H), 1.88 1.74 (m, 4 H), 1.49 1.25 (m, 12 H), 0.91 (t, J = 13.6 Hz, 6

H).


8.8 Hz, 4 H), 7.01 (d, J = 8.3 Hz, 4 H), 6.99 (s, 2 H), 6.94 (d, J = 12.8 Hz, 4 H), 4.07 3.99

(m, 8 H), 1.88 1.77 (m, 8 H), 1.55 1.45 (m, 8 H), 1.42 1.33 (m, 16 H), 0.97 0.89

(t, J = 6.8 Hz, 12 H).


2.0 Hz, 4 H), 7.02 (d, J = 2.2 Hz, 4 H), 6.99 (d, J = 2.2 Hz, 4 H), 6.95 (s, 2 H), 4.08 4.03

(m, 8 H), 1.88 1.79 (m, 8 H), 1.45 1.32 (m, 24 H), 0.98 0.96 (m, 12 H).

B1: 1H NMR (400MHz, CDCl3) δ (ppm) : 7.83 (d, J = 8.8 Hz, 4 H), 7.67 (d, J = 8.9 Hz, 4 H),

7.01 6.95 (m, 8 H), 4.07 3.98 (m, 8 H), 1.88 - 1.78 (m, 8 H), 1.55 1.43 (m, 8 H),

1.42 1.35 (m, 16 H), 0.98 0.91 (m, 12 H).

13C NMR (100 MHz, CDCl3) δ (ppm): 161.08, 160.39, 160.11, 147.25, 144.80, 132.38,

132.33, 124.44, 123.29, 113.94, 113.79, 68.13, 67.99, 31.62, 31.58, 29.23, 29.21,

25.78, 25.76, 22.65, 22.63, 14.07, 14.06.

4. Synthesis of CaO2 nanoparticles

CaO2 nanoparticles were prepared according to the previous work reported by

Khodaveisi et al.2. CaCl2 (3 g) were dissolved in 30 mL ultrapure water, followed by

adding 15 mL NH3H2O (1 M) and 94 mL PEG200. Then 15 mL H2O2 (30%) was gradually

added to the mixture at the rate of three drops per min. The mixture was further

stirred for another 1 h. To precipitate the prepared nanoparticles, pH value of the

mixture was adjusted to about 11 using NaOH (0.1 M). Finally, CaO2 nanoparticles

were obtained by centrifugation at 10000 rpm for 5 min and washed three times with

NaOH solution and ultrapure water.

5. Synthesis of liposomes

DPPC (5 mg), cholesterol (1.8 mg) and DSPE-PEG5000 (3.2 mg) were mixed with B1 (1

mg) and dissolved in chloroform. Then 200 L CaO2 nanoparticles (10 mg/mL)

dispersed in ethanol were added. The mixture was dried via a rotary evaporator under

a decreased pressure to form a lipid film. 10 mL NH4HCO3 (2.7 M) were added into the

lipid film, following the continuous sonication (360 W) at room temperature for 30 s.

The suspension was centrifuged at 10000 rpm for 10 min, and washed with three

times with ultrapure water to remove the unencapsulated NH4HCO3. The control

groups of liposomes were prepared according to a similar procedure without adding

B1, CaO2 nanoparticles or NH4HCO3. Finally, the liposomes were dispersed in PBS

buffer.

6. Evaluation of 1O2 generation of liposomes

1,3-diphenylisobenzofuran (DPBF) was used as a 1O2 indicator. The air-saturated

liposomes (184 g/mL) dispersed in PBS buffer containing 10 L DPBF (500 M) was

prepared. The solution in the hypoxia environment was produced by bubbling with

Argon. The solution was irradiated under a NIR 730 nm laser (500 mW/cm2) in an

interval of 1 min at 37 C.

7. Cell culture

HeLa cell lines were provided by the Institute of Biochemistry and Cell Biology, SIBS,

CAS (China). The cells were grown in DMEM (Dulbecco's modified Eagle’s medium)

supplemented with 10% FBS (fetal bovine serum), 100 mg/mL streptomycin and 100

U/mL penicillin at 37 °C with 5% CO2.

8. Cellular uptake of liposomes

HeLa cells were incubated in DMEM (high glucose) medium containing 10% FBS at 37

C in a humidified atmosphere containing 5% CO2. HeLa cells were seeded in the

confocal dishes at a density of 105 cells/dish. After 24 h incubation, cells were attached

and treated with free medium containing liposomes (80 g/mL) for another 2 h.

9. Cellular ROS generation

Cells were cultured with different groups of liposomes in normoxic or hypoxic

condition for 2 h, followed by the treatment of the commercial ROS indicator, DCFH-

DA (10 M) for another 2 h. Then cells were irradiated by the laser (730 nm 500

mW/cm2) for 5 min. The fluorescence from DCF was collected (ex = 488 nm, em = 500

– 540 nm). Cells were cultured with an atmosphere of 21% O2 and 5% O2 at 37 C to

mimic the normoxic and hypoxic environment, respectively.

10. Calcein-AM /PI assays

The viability of cells incubated with liposomes was evaluated via Calcein-

AM/propidium iodide (PI) staining. Cells were cultured at 100% humidity 37 °C with 5%

CO2 for 24 h. Liposomes (80 g/mL) were added to the medium and cells were

incubated for another 2 h under 5% or 21% O2. Then the cells were treated with PI (5

µL) and Calcein-AM (10 µL) in dark for 10 min and then irradiated with a 730 nm laser

(500 mW/cm2) for 5 min. The images were obtained by the confocal microscopy in

green channel for Calcein-AM (λex = 488 nm, λem = 500 – 560 nm) and red channel for

PI (λex = 488 nm, λem = 600 – 680 nm) after 1 h.

11. Cell viability

The cytotoxicity of liposomes towards HeLa cells was assessed by the methyl thiazolyl

tetrazolium (MTT) assay. HeLa cells in log phase were seeded into a 96-well cell-

culture plate at 1 × 104/well, followed by incubation at 37 °C and 5% CO2 atmosphere

for 24 h. The liposomes at concentrations of 20, 40, 80, 120, 160 g/mL was added to

the wells. To evaluate the dark cell viability, the cells were incubated in dark at 37 °C

and 5% CO2 atmosphere for 24 h. To assess the phototoxicity, cells were irradiated

with a 730 nm laser (500 mW/cm2) for 5 min and then further incubated for 24 h. 20

mL MTT solution (5 mg mL-1) was added to each well of the 96-well assay plate, and

the solution was incubated for another 3 h under the same condition. The OD570

(absorbance value) of each well referenced at 690 nm were measured using a Tecan

Infinite M200 monochromator based multifunction microplate reader. The following

formula was used to calculate the viability of cell growth: viability (%) = [(mean of

absorbance value of treatment group)/(mean absorbance value of control)]×100%.

12. Annexin V-FTIC/PI assays

HeLa cells were seeded in the 6-well plates and incubated at 100% humidity 37 °C with

5% CO2 for 24 h. Liposomes (80 g/mL) were added to the medium and cells were

incubated for another 2 h under 5% or 21% O2. Then cells were irradiated with a 730

nm laser (500 mW/cm2) for 5 min and further incubated in dark for 24 h under 5% or

21% O2. Finally, cells were stained with Annexin-FITC/PI for 15 min and digested using

trypsin and collected in centrifuge tubes. Cells were then analyzed by flow cytometry.

13. Animal model

The female athymic nude mice were purchased from Comparative Medicine Center of

Yangzhou University (Permit number: 201825343) with relevant laws and guidelines.

Mice bearing tumor models were established by subcutaneous injection of HeLa cells

(about 106 per mouse) into the flanks of the mice. When the tumor volume reached

to about 100 mm3, we began to conduct the relevant experiments.

14. In vivo therapy

Mice were divided into 7 groups and injected with CaO2/B1 lipo (800 g/mL, 100 L),

CaO2/B1/NH4HCO3 lipo (800 g/mL, 100 L), or PBS. After 4 h, the groups treated with

liposomes was injected with NAC (50 mM) or not and exposed under irradiation with

a 730 nm laser (100 mW/cm2) for 5 min or not. The tumor volume and body weight

were estimated every 2 days for 14 days. The tumor volume was calculated according

to the follow formula: V = length×width2/2. The ratio tumor volume V/V0 was

measured for investigation. After 14 days, the mice were sacrificed. Tumors and major

organs were collected and fixed using 4% formalin solution for further H&E staining

and immunofluorescence staining of CA9 and HIF-1.

1 10 100 10000

20

40

60

80

100

Si

ze d

istri

butio

n (n

umbe

r%)

Size (nm)1 10 100 1000 10000

0

20

40

60

80

100

Size

dis

tribu

tion

(num

ber%

)

Size (nm)

a b

Fig. S1 The DLS results of CaO2 nanoparticles and CaO2/B1/NH4HCO3 lipo.

200 nm 200 nm

a b

Fig. S2 TEM images of CaO2/B1/NH4HCO3 lipo prepared under the sonication power of

600 W (a) and 180 W (b).

Since the size of liposomes is associated with sonication power, we also prepared

CaO2/B1/NH4HCO3 lipo under sonication power of 180 W and 600 W, and

characterized the morphology of the liposomes. As seen in Fig. S2, when the power

was 600 W, the lipid membrane and CaO2 nanoparticles were observed in the TEM

image, which may be ascribed to the instability of liposomes. When the power was

decreased to 180 W, CaO2/B1/NH4HCO3 lipo were aggregated and difficult to be well

dispersed in PBS buffer. Thus we chose the liposomes prepared under sonication

power of 360 W for further investigation.

810 820 830 840 8500.0

4.0x103

8.0x103

1.2x104 CaO2/B1/NH4HCO3 lipo

PL In

tens

ity (a

.u.)

Wavelength (nm)200 400 600 800

0.0

0.2

0.4

0.6

0.8

1.0Ab

s.

Wavelength (nm)

CaO2/B1/NH4HCO3 lipo

a b

Fig. S3 The absorption and emission spectra of CaO2/B1/NH4HCO3 lipo.

37 C 45 C

200 m

Fig. S4 Bright images of PBS buffer containing NH4HCO3 lipo at 37 and 45 C.

Fig. S5 (a) Schematic diagram of the evaluation of photothermal effects; (b)

temperature changes of NH4HCO3 lipo or CaO2/NH4HCO3 lipo (246 g/mL) under

different irradiation time; (c) temperature of NH4HCO3 lipo or CaO2/NH4HCO3 lipo under

different distance in the longitudinal axis away from the laser beam after irradiation for 5

min. The irradiation power is 192 mW. Beam area is 0.38 cm2.

To investigate the heat diffusion, we tested the point temperature in the cell

containing the liposomes after irradiation for 5 min (Fig. S5c). The d represents the

distance in the longitudinal axis away from the laser beam. The temperature varied

from ~24 to ~23 C with the distance increased. The small changes of temperature

could be ascribed to the absence of photothermal molecule B1 in the liposomes.

0 5 10 15 204

5

6

7

8

9

10

11

pH v

alue

Time (min)

123 g/mL 184 g/mL 246 g/mL

Fig. S6 pH values of CaO2/B1/NH4HCO3 lipo with different concentrations under irradiation

for 20 min.

400 600 8000.0

0.4

0.8

1.2

1.6

2.0

Abs.

Wavelength (nm)

0 min 1 min 2 min 3 min 4 min 5 min 6 min 7 min

400 600 8000.0

0.4

0.8

1.2

1.6

2.0

Abs.

Wavelength (nm)

0 min 1 min 2 min 3 min 4 min 5 min 6 min 7 min

0 2 4 6

0.0

0.2

0.4

0.6

0.8

A

Time (min)

CaO2/B1 lipo CaO2/B1/NH4HCO3 lipo

a b c

Fig. S7 Absorption spectra of the mixture containing DPBF and CaO2/B1 lipo (a),

CaO2/B1/NH4HCO3 lipo (b) in air at different irradiation time in air at 37 C; (c) A of DPBF

at 427 nm which are obtained from figure a and b. A = At A0, At was the absorbance of DPBF

at 427 nm at different irradiation time and A0 was the absorption without irradiation.

21% O2

5% O2

a b c

30 m

Fig. S8 ROS generation in blank cells (a), cells incubated with NH4HCO3 lipo (b) or

CaO2/NH4HCO3 lipo (c) (80 g/mL) under 21% or 5% oxygen level and then exposed under

730 nm laser irradiation (500 mW/cm2) for 5 min.

Calcein-AM

PI

Overlay

a b c d e

20 m

Fig. S9 Calcein-AM/PI assays of blank cells (a), cells incubated with NH4HCO3 lipo (b),

CaO2/NH4HCO3 lipo (c), CaO2/B1 lipo (d), CaO2/B1/NH4HCO3 lipo (e) (80 g/mL) under

21% oxygen level and then exposed under 730 nm laser irradiation (500 mW/cm2) for 5 min.

Calcein-AM

PI

Overlay

a b c

20 m

Fig. S10 Calcein-AM/PI assays of blank cells (a), cells incubated with NH4HCO3 lipo (b)

or CaO2/NH4HCO3 lipo (c) (80 g/mL) under 5% oxygen level and then exposed under 730

nm laser irradiation (500 mW/cm2) for 5 min.

Calcein-AM PI Overlayb c

21% O2

5% O2

a

b

20 m

Fig. S11 Calcein-AM/PI assays of cells incubated with CaO2/B1/NH4HCO3 lipo (80

g/mL) and further treated with NAC (5 mM) under 730 nm laser irradiation (500 mW/cm2)

for 5 min under 21% (a) and 5% (b) oxygen level.

0 20 40 80 120 1600

20

40

60

80

100

120

Cell

Rela

tive

Viab

ility

(%)

Concentration (μg/mL)

Dark Light

0 20 40 80 120 1600

20

40

60

80

100

120

Cell

Rela

tive

Viab

ility

(%)


Dark Light

0 20 40 80 120 1600

20

40

60

80

100

120

Cell

Rela

tive

Viab

ility

(%)


Dark Light

0 20 40 80 120 1600

20

40

60

80

100

120

Cell

Rela

tive

Viab

ility

(%)


Dark Light

21% O2

5% O2

a b

c d

Fig. S12 MTT assays of cells incubated with (a, c) NH4HCO3 lipo or CaO2/NH4HCO3 lipo

(b, d) under 21% and 5% oxygen atmosphere with and without irradiation.

5% O2

21% O2

CaO2/ NH4HCO3 lipo

92.4% 3.87%

1.83% 1.9%

NH4HCO3 lipo

94.2% 4.28%

0.97%0.56%

CaO2 /B1 lipo

2.27%

0.99%1.00%

95.7%


95.3% 3.08%

1.03%0.56%

81.6% 15.2%

2.92%0.26%

89.5% 5.92%

3.84%0.67%

85.5% 9.63%

4.36%4.36%

84.1% 4.77%

4.65%6.41%

Fig. S13 Flow cytometry results of cells incubated with different liposomes (80 g/mL)

under 21% and 5% oxygen atmosphere without irradiation.

76.9% 17.5%

5.04%0.37%

74.3% 11.5%

10.5%3.67%

CaO2/ NH4HCO3 lipo

92% 5.11%

2.06%0.8%

NH4HCO3 lipo

4.86% 2.73%

86.7% 5.74%

5% O2

21% O2

Fig. S14 Flow cytometry results of cells incubated with CaO2/NH4HCO3 lipo or NH4HCO3

lipo (80 g/mL) under 21% and 5% oxygen atmosphere with irradiation (730 nm 500

mW/cm2).

0 5 10 15 20 25

8x107

9x107

1x108

1x108

Tu

mor

fluo

resc

ence

(a.u

.)

Time (h)Fig. S15 The tumorous fluorescence monitored at different time.

4.00 e7

8.00 e7

Fig. S16 Fluorescence images of the main organs of the mouse after 4 h injection of

CaO2/B1/NH4HCO3 lipo. From left to right is tumor, heart, liver, spleen, lung and

kidney.

0 100 200 300 400 500

0

2

4

6

8

10

0 100 200 300 400 500

0

2

4

6

8

10

0 100 200 300 400 500

0

2

4

6

8

10

0 100 200 300 400 500

0

2

4

6

8

10

0 100 200 300 400 500

0

2

4

6

8

10

+CaO2/B1/NH4HCO3 lipo +hT

(C)

Time (s)

T (

C)

Time (s)

+CaO2/B1/NH4HCO3 lipo + NAC h +CaO2/B1 lipo +h

T (

C)

Time (s)

T (

C)

Time (s)

T (

C)

Time (s)

+PBS h

Fig. S17 Temperature changes of tumors with different treatments under laser

irradiation for 7 min (730 nm, 100 mW/cm2).

PBS

PBS h

CaO2/B1 lipo

CaO2/B1 lipo +h

CaO2/B1/NH4HCO3 + NAC h


CaO2/B1/NH4HCO3 lipo + h

0 d 2 d 4 d 8 d 14 d

Fig. S18 Photographs of mice treated with different groups during therapy procedure.

Fig. S19 The H&E stained slices harvested from the main organs of mice after different

treatments.

Fig. S20 Immunofluorescence staining of HIF-1 and CA9 of tumor slices with different

treatments.

1H NMR spectrum of compound 1

-2.0-1.00.01.02.03.04.05.06.07.08.09.010.011.0f1 (ppm)

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

6.12

12.6

3

4.28

4.12

4.03

1.15

2.03

1.11

2.00

0.91

1.35

1.80

1.81

1.83

3.98

4.00

4.02

4.03

4.05

6.91

6.93

6.95

6.97

7.41

7.57

7.75

8.01

8.03

C6H13O

O

OC6H13


-1.5-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.5f1 (ppm)

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000

12.3

2

16.3

88.

16

8.02

8.19

4.06

1.95

4.00

4.05

4.00

0.92

0.93

0.95

1.35

1.39

1.80

1.81

1.83

1.85

1.86

4.01

4.02

4.02

4.04

4.04

4.05

6.93

6.95

6.99

7.01

7.02

7.83

7.85

8.00

8.02

NH

O

O

N

N

O

OC6H13

C6H13 C6H13

C6H13


-1.5-0.50.51.52.53.54.55.56.57.58.59.510.5f1 (ppm)

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

12.3

9

24.5

8

8.12

8.29

2.16

3.93

4.21

4.04

3.86

0.95

0.95

1.38

1.38

1.39

1.84

1.86

6.95

6.99

6.99

7.00

7.01

7.01

7.02

8.06

8.06

8.08

8.08

N

O

O

N

N

O

OC6H13

C6H13 C6H13

C6H13

BF F

1H NMR spectrum of B1

N

O

O

N

N

O

OC6H13

C6H13 C6H13

C6H13

BI I

F F

-1.5-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.5f1 (ppm)

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

12.0

6

15.8

47.

86

7.93

8.12

8.10

4.06

4.00

0.89

0.90

0.92

0.94

0.95

0.96

1.36

1.40

1.79

1.81

1.83

1.85

1.87

1.88

4.00

4.02

4.03

4.05

4.06

6.95

6.97

6.98

6.99

7.66

7.68

7.82

7.84

13C NMR spectrum of B1

N

O

O

N

N

O

OC6H13

C6H13 C6H13

C6H13

BI I

F F

-20-100102030405060708090110130150170190210f1 (ppm)

0

5E+07

1E+08

2E+08

2E+08

2E+08

3E+08

4E+08

4E+08

4E+08

MALIDI-TOF-MS spectrum of B1

800 1000 1200 1400 16000

1000

2000

In

tens

ity (a

.u.)

m/z

1149.331

Reference

1. Zhao M., Xu Y., Xie M., Zou L., Wang Z., Liu S., Zhao Q., Adv. Healthcare Mater.,

2018, 18, 1800606.

2. Khodaveisi J., Banejad H., Afkhami A., Olyaie E., Lashgari S., Dashti R. J. Hazard

Mater., 2011, 192, 1437.

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