S1

Supporting Information

A New Polymer Nanoprobe Based on Chemiluminescence Resonance

Energy Transfer for Ultrasensitive Imaging of Intrinsic

Superoxide Anion in Mice

Ping Li, Lu Liu, Haibin Xiao, Wei Zhang, Lulin Wang, and Bo Tang*

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of

Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano

Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals,

Shandong Normal University, Jinan 250014, P. R. China

*E -mail: tangb@sdnu.edu.cn

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Table of Contents

page

1. Materials and Instruments………………………………………………………………………………………...S3

2. Experimental sections ……………………..……………………………………………………………………..S4

3. The synthesis of PCLA-O2•−

………………………………………………………………….…………………..S6

4. Luminescence mechanism of PCLA-O2•−

………………………………………...………..…………...............S11

5. FT-IR spectra of PFBT, 5 and 6...………………………………………………………………………………S11

6. CL responses of PFBT and CHO-CLA mixtures to O2•−

………...…………………...…...…………….……...S11

7. The selectivity of PCLA-O2•−

………………………………………..………………………………………….S12

8. Effects of pH on CL responses of PCLA-O2•−

to O2•−

..…………………………………...……………….……S13

9. Effects of mice serum on CL of PCLA-O2•−

in response to O2•−

……..……………………………..…….....…S14

10. The stability of PCLA-O2•−

over time…………….…….……………………………….…………….….…...S14

11. CL responses of PCLA-O2•−

in MCF-10A, 4T1 extracts and macrophages extracts………...……………..…S15

12. Cytotoxicity assay and in vivo toxicity study……..……………………………………….……………….….S15

13. The CL images of endogenous O2•−

in the LPS-induced inflammatory in mice over time……………………S16

References..………………………………………………………………………………………………………...S17

S3

1. Materials and Instruments

Materials. All reagents were purchased commercially and used without further purification.

2-amino-5-bromopyrazine, 4-formylphenylboronic acid, methylglyoxal, 2,7-dibromofluorene, 1,6-dibromohexane,

tetrabutylammonium bromide (Bu4NBr), N-boc-ethylenediamine, bis(pinacolato)diboron,

4,7-dibromo-2,1,3-benzothiadiazole, trifluoroacetic acid (TFA), trimethylamine, the catalysts Pd(PPh3)4 and

Pd(dppf)Cl2 were from Adamas-beta and phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS),

superoxide dismutase (SOD) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from

Sigma-Aldrich. 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron) was purchased from Shanghai

Reagent Co. Ltd. (Shanghai, China). The mouse mammary carcinoma 4T1 cells and macrophages were purchased

from Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Instruments. NMR spectra were recorded mainly on Bruker NMR spectrometers operating at 400 MHz for 1H and

at 100 MHz for 13

C. The mass spectra were obtained by Bruker maXis ultra-high resolution-TOF MS system. The

fluorescence spectra measurements were performed using FLS-920 Edinburgh fluorescence spectrometer. High

resolution transmission electron microscopy (HRTEM) was carried out on a JEM-2100 electron microscope.

Absorption spectra were measured on a TU-1900 UV-vis spectrophotometer (Beijing Purkinje General Instrument

Co., Ltd.). Absorbance was measured in a microplate reader (RT 6000, Rayto, USA) in the MTT assay. Cell

extracts were prepared by using a BILON92-IIL ultrasonic disintegrator (Shanghai Bilon Materials Inc.). GPC

measurements for determination of molecular weights and the polydispersity index (PDI) of the polymers were

performed in tetrahydrofuran (THF) using a Waters 515 refractive-index detector. Polystyrene standards purchased

from Polymer Standard Service were used for calibration. Dynamic light scattering (DLS) measurements and

Zpotential were performed on a Malvern zeta sizer Nano-ZS90. FTIR spectra were collected on a Nicolet Impact 410

FTIR spectrometer in the range of 400-4000 cm-1

. CL imaging was performed by an IVIS Lumina II in vivo

S4

imaging system.

2. Experimental sections

Detection of the limit of detection (LOD)[1]

According to the reference, the limit of detection (LOD) was calculated based on IUPAC utilizing the equation

LOD=3S0/K, where S0 is the standard deviation of blank measurements (n=9) and K is the slope of linear

regression equation.

Optical responses of PCLA-O2.- toward various reactive species

Reactive species were as follows. Superoxide (O2•−

) was delivered from potassium oxide (KO2) in DMSO solution,

and absorption spectrum was measured to quantify the concentration of O2•−

by a TU-1900 UV-vis

spectrophotometer. Hydroxyl radical (·OH) was generated from Fenton reaction (Fe2+

+H2O2→Fe3+

+·OH+OH-).

H2O2, tert-butyl hydroperoxide (TBHP), and hypochlorite (NaOCl) were obtained from directly diluting

commercially available 30%, 70%, and 10% aqueous solutions, respectively. Ascorbic acid and glutathion (GSH)

stock solution were prepared from commercially respective solid. Nitric oxide (NO) was used from stock solution

prepared by sodium nitroprusside. Singlet oxygen (1O2) was produced from the ClO

-/H2O2 system. Peroxynitrite

(ONOO-) was prepared from stock solution, and absorbance was measured at 302 nm to quantify the concentration

of ONOO-. In selectivity of PCLA-O2

•− experiments, PCLA-O2

•− (0.45 mg/ml in PBS containing 10% THF, 100 µL)

was placed in a white 96-well plate. After addition of different reactive species, the CL (λem=570±10 nm) images

were obtained by an IVIS Lumina II in vivo imaging system with open filter. The CL intensities were output from

the white 96-well plate region.

DL of PCLA-O2•−

and CHO-CLA in response to O2•−

For DL of PCLA-O2•−

and CHO-CLA in response to O2•−

, the CL intensities of the PCLA-O2•−

(0.45 mg/mL) and

CHO-CLA (0.0042 M) solution in PBS buffer (10 mM, pH = 7.4, 10% THF) were measured after the addition of

S5

O2•−

(0.1 µM and 0.9 µM, respectively). At intervals of about 0.7 min, the CL images of PCLA-O2•−

(λem=570±10

nm) and CHO-CLA (λem=520±10 nm) solution were obtained by an IVIS Lumina II in vivo imaging system with

open filter. The intensities were output from a white 96-well plate region and plotted as a function of time.

Cell extracts analysis

The concentration of counted cells was ~ 1.0×106 cells mL

-1. The cells without PMA-stimulated were used as the

control group. In addition, the other three groups were incubated with PMA (1.0 µg/mL) for 1 h. One group of

PMA-stimulated cells were incubated with Tiron (10 µM) for 1h. Subsequently, the four groups of cells were

suspended in a volume of PBS and disrupted for 10 min in a VC 130PB ultrasonic disintegrator (<4°C). One group

of PMA-stimulated cell extracts were incubated with SOD (200 U) for 30 min. Then, the four groups of broken cell

suspension were centrifuged at 4000 rpm for 10 min and the pellet discarded. Finally, with addition of PCLA-O2•−

(0.45 mg/mL), the CL (λem=570±10 nm) images of the four groups were acquired by an IVIS Lumina II in vivo

imaging system with open filter. The similar disrupted process was performed in MCF-10A and 4T1 cells.

LPS-induced inflammation model in Mice

All 8-10-week-old KM mice were purchased from Shandong University Laboratory Animal Center. All the animal

experiments were carried out in accordance with the relevant laws and guidelines issued by the Ethical Committee

of Shandong University. In LPS-induced inflammation model, three groups of mice were treated with four different

conditions. (I) LPS (400 µL, 2.5 mg/ml in saline) was injected into peritoneal cavity of mice. 4 h later, PCLA-O2•−

(0.068 mg) was injected. (II) LPS (400 µL, 2.5 mg/ml in saline) was administrated into abdomen. 3 h later, Tiron

(400 µL, 10 µM) was injected. 1 h later, PCLA-O2•−

(0.068 mg) was injected. (III) Saline was injected into the

abdomen, followed by an intraperitoneal injection PCLA-O2•−

(0.068 mg) 4 h later. (IV) The control: only saline

was injected. Then, mice were anesthetized with 4% chloral hydrate (3 mL/kg) by intraperitoneal injection and the

hair and skin on the ventral surface of the abdomen was removed. Then，PCLA-O2.-

(0.068 mg) was given to the

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mice intraperitoneally. CL (λem=570±10 nm) images were acquired by an IVIS Lumina II in vivo imaging system

with open filter within 25 min.

MTT Assay and in vivo toxicity experiments

To investigate PCLA-O2•−

cytotoxicity, MTT assay were carried out in macrophages. Macrophages were seeded at a

density of 106

cells/well in a 96 well-plate and incubated at 37 °C in a 5% CO2/95% air for 24 h before adding

PCLA-O2

•−. Then upon different PCLA-O2

•− concentrations of 0.45, 0.90, 1.30 and 2.25 mg/mL, macrophages were

incubated for 12 h. The culture medium was removed, and MTT solution (0.5 mg /mL) was then added to each well.

After 4 h, the remaining MTT solution was removed, and DMSO (150 µL) was added to each well to dissolve the

formazan crystals. Absorbance was measured at 490 nm in a microplate reader. For in vivo toxicity study, the KM

mice in two groups were intraperitoneally (i.p.) administrated PCLA-O2•−

containing 10% THF with experimental

concentration 0.068 and even more higher concentration 0.136 mg , and the third group were injected 0.9% NaCl

aqueous solution as the nontoxic control. Clinical symptoms such as the weight, hair loss, scab, vomiting and

diarrhea were observed for 2 weeks after injection with PCLA-O2•−

. And the clinical symptoms of three groups

mice were recorded every day.

3. Synthesis of probes (PCLA-O2•−

)

Compounds A and CHO-CLA were synthesized according to the previous literature. [2]

Scheme S1. Synthesis of A and CHO-CLA; i) Na2CO3, Pd(PPh3)4, toluene, ethanol, 85 °C, 12 h; ii) HCl, ethanol,

78 °C, 6h. (3 mmol, 0.45 g)

The synthesis of A

In a 100 mL flask, 2-amino-5-bromopyrazine (3 mmol, 0.51 g) and 4-formylphenylboronic acid were dissolved in

toluene (20 mL) and ethanol (10 mL), and then Na2CO3 (2 M, 6 mL) was added. After degassing, Pd(PPh3)4 (0.044

S7

mmol, 0.05 g) was added. The reactants were stirred at 85 °C for 12 h. The residue was purified by column

chromatography with methanol/ dichloromethane (1:10) as eluent and yellow product was obtained. Yield: 0.39 g

(65 %). 1HNMR (400 MHz, DMSO) 6.85 (s, 2H), 7.94 (s, 2H), 8.03 (s, 1H), 8.14(s, 2H), 8.65 (s, 1H), 10.00 (s, 1H).

13CNMR (100 MHz, DMSO) 125.33, 130.52, 131.89, 135.31, 137.76, 140.69, 143.24, 155.98, 192.97. HRMS date,

m/z calcd for C11H9N3O [M-H-]: 198.0661, found 198.0907.

The synthesis of CHO-CLA

A mixture of A (0.10 g, 0.50 mmol) and methylglyoxal (0.238 g, 3.3 mmol) in 8 ml ethanol containing

hydrochloric acid (200 µL) were refluxed at 78 °C under nitrogen atmosphere for 6 h. Solvents were concentrated

in vacuum, and the residue was purified by thin layer chromatography of silica gel GF254 with

dichloromethane/methyl alcohol (5:1) as eluent and light yellow product CHO-CLA was obtained. Yield: 0.038 g

(30%). 1HNMR (400 MHz, CH3OH) 2.59 (s, 3H), 5.48 (s, 1H), 7.63 (d, J=8.0 Hz 2H), 8.08 (d, J=7.8 Hz 2H), 8.78

(s, 1H), 9.20 (s, 1H). 13

CNMR (100 MHz, CH3OH) 8.90, 111.47, 121.67, 126.36, 126.95, 127.25, 133.82, 134.88,

138.07, 139.91, 140.14. HRMS date, m/z calcd for C14H11N3O2 [M-H-]: 252.0767, found 252.0702.

Compound 1 and 2 was synthesized as previous report.[3]

The synthesis of 1, 2, 3, PFBT, 5, 6[4]

and probe PCLA-O2.-

S8

S9

Scheme S2. The synthesis of 1, 2, 3, PFBT, 5 and 6; Reaction conditions: a) 1,6-dibromohexane, Bu4NBr, KOH,

75 °C, 25 min; b) Pd(dppf)Cl2, KOAc, dioxane, 85 °C, 12 h; c) NaOH, acetonitrile, 80 °C, 3 h; d) Pd(PPh3)4,

K2CO3, toluene/H2O, 90 °C, 24 h; e) (i) THF/H2O, NMe3, 25 °C, 24 h . (ii) dichloromethane, TFA, 25 °C, 1 h; f)

ethanol, 25 °C, 6 h.

The synthesis of 3

2,7-bis[9,9’-bis(6”-bromohexyl)-fluorenyl]-4,4,5,5-tetramethyl-[1.3.2]dioxaboolane (0.30 g, 0.37 mmol),

N-boc-ethylenediamine (0.253 g, 1.58 mmol), NaOH (0.03 g, 0.75 mmol) were placed in a 25-mL round-bottom

flask. Acetonitrile (6 mL) was added to the flask and the reaction vessel was degassed. The resulting mixture was

heated at 80 °C for 3 h. The reaction solution was purified by thin layer chromatography of silica gel GF254 with

ethyl acetate/methyl alcohol (3:1) as eluent and white product 3 was obtained. Yield: 0.164 g (55%). 1HNMR (400

MHz, DMSO) 0.80-1.23 (m, 16H), 1.34 (s, 18H), 1.80-2.50 (m, 12H), 2.95 (s, 4H), 6.64 (s, 2H), 7.40-7.80 (m, 6H).

13CNMR (100 MHz, CDCl3) 23.48, 26.89, 28.16, 29.58, 29.69, 40.13, 41.73, 49.00, 49.30, 55.52, 78.82, 121.14,

121.41, 126.01, 130.15, 138.96, 152.27, 156.10. HRMS date, m/z calcd for C39H60N4O4Br2 [M+H] +

: 809.3037,

found 809.2870.

The synthesis of PFBT

In a 50 mL round-bottom flask, monomer 2 (0.566 g, 0.7 mmol), 3 (0.75 g, 1 mmol), and

4,7-dibromo-2,1,3-benzothiadiazole (0.087 g, 0.3 mmol) were dissolved in toluene (10 mL), and then Bu4NBr (0.1

mmol, 0.032 g) and Na2CO3 (2 M, 6 mL) were added. The mixture was degassed and refilled with Ar gas (repeated

3 times) before and after addition of Pd(PPh3)4 (0.17 mmol, 0.20 g). The reactants were stirred at 90 °C for 24

hours and then cooled to the room temperature. The mixture was poured into methanol (100 mL). The polymer was

washed with methanol to remove monomers, small oligomers, and inorganic salts and then dried under vacuum for

48 h to afford precursor (0.443 g, 65%) as a bright-yellow powder. 1HNMR (400 MHz, CDCl3) 0.70-1.20 (m, 36 H),

S10

1.25 (s, 18H), 1.35-1.80 (m, 28H), 2.91 (s, 4H), 3.51 (s, 4H), 3.58 (s, 8H), 5.41 (m, 2H), 6.75-8.20 (m, 20H).

13CNMR (100 MHz, CDCl3) 22.61, 25.69, 26.73, 27.46, 27.83, 28.68, 30.90, 32.96, 36.08, 39.13, 48.06, 48.43,

52.76, 54.61, 77.84, 125.09, 126.56, 127.05, 127.93, 129.22, 131.30, 138.00, 151.35, 152.98, 153.36, 155.03. GPC

(tetrahydrofuran, polystyrene standard), Mw: 134876 g/mol; Mn: 68382 g/mol; PDI: 1.9723. The average degree of

polymerization n=68.

The synthesis of 5

In a 100 ml round-bottom flask, neutral precursor polymer PFBT (0.221 g) was dissolve in 10 ml THF at -78 °C by

stirring. After degassing, the condensed trimethylamine (2 mL) was added. The mixture reacted at room

temperature for 12 h. The precipitate was redissolved by addition of water (10 mL). After the mixture was cooled

down to -78 °C, more trimethylamine (2 mL) was added and the mixture was stirred for 12 h at room temperature.

Subsequently, most of the solvent was removed using the vacuum pump (20 mm Hg). Then a mixture of cationic

polymers and TFA (0.5 mL) in dichloromethane (10 mL) reacted at 25 °C under nitrogen atmosphere for 1 h in

darkness. The reaction mixture was washed with 10% NaOH water solution three times and added the excessive

water. The solvent was extracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4. The organic

phase was removed to obtain the polymer product 5 (0.132 g, 60%) under reduced pressure. 1HNMR (400 MHz,

CDCl3) 1.25-1.40 (m, 40H), 1.80-2.10 (m, 32H), 3.58 (s, 8H), 3.73 (s, 36H), 7.10-8.01 (m, 20H).

The synthesis of 6

Polymer product 5 (0.132 g), CHO-CLA (0.068 g) were placed in 10 mL of ethanol, and the mixture was stirred at

25 °C under nitrogen atmosphere for 12 h. The resulting solution was centrifuged and the obtained solid was

washed to remove CHO-CLA.

The synthesis of probe (PCLA-O2•−

)

The probes were synthesized using nano-precipitation. The solution of 6 (0.0045 g) in 1 mL of THF is rapidly

S11

added to an excess of water (8 mL) under continue ultrasonification. After sonication for additional 2min, THF was

evaporated under nitrogen atmosphere.

4. Luminescence mechanism of PCLA-O2•−

5. FT-IR spectra of PFBT, 5 and 6

Figure S1 1 1 1 FT-IR spectra of PFBT, 5 and 6.

6. CL responses of PFBT and CHO-CLA mixtures to O2•−

To assess the efficiency of CRET between CLA and PFBT by covalent conjugation, a control experiment was

performed in a mixed aqueous solution of CHO-CLA to PFBT. As can be seen in Figure S2, CL responses to O2•−

at

570 nm basically kept unchanged, even with increasing number of CHO-CLA level. This result demonstrates a part

S12

of small molecule CHO-CLA may indeed disperse in a miscible solvent, which cause low CRET efficiency. The

result testifies that covalent linkage of CLA to PFBT in PCLA-O2•−

is essential in avoiding the dispersive problem

of CHO-CLA. Meanwhile, covalent linkage can powerfully control close distance of donor and acceptor, thereby, a

higher efficiency CRET between CLA and PFBT can be realized.

Figure S2 The CL responses (left: λem=520±10 nm; right: λem=570±10 nm) of the physical mixture of PFBT and

CHO-CLA to O2•−

(0.90 µM) in 10 mM PBS buffer (pH 7.4, 10% THF). [CHO-CLA]=0.70, 1.4, 2.1, 2.8, 3.5, 4.2

mM, respectively. n [PFBT] : [CHO-CLA]=1:2, 1:4, 1:6, 1:8, 1:10, 1:12. The average degree of polymerization

n=68.

7. The selectivity of PCLA-O2•−

In order to examine whether PCLA-O2•−

could specifically monitor O2•−

with complexity of the intracellular

environment, we studied the interference of the bio-relevant substances, including H2O2, 1O2, NO, ascorbic acid,

ClO−, TBHP, ·OH, GSH, ONOO

−. Figure S3 showed PCLA-O2

•− CL was unaffected by various reactive species.

S13

Figure S3 The CL ratios of 0.45mg/ml PCLA-O2•−

to various reactive species in 10 mM PBS buffer (pH 7.4, 10%

THF). Red bars represent the addition of different reactive species (100 µM for GSH; 1 mM for H2O2, Vc; 200 µM

for ClO−, TBHP, ·OH,

1O2, NO; 130 µM for ONOO

−; 1.0 µM O2

•−). Relative CL intensity (CL/CL0, in which CL

and CL0 represent the emission CL intensity in the presence and the absence of various reactive species,

respectively).

8. Effects of pH on CL responses of PCLA-O2•−

to O2•−

Because pH of the buffer solution plays an important role in the monitoring O2•−

, we investigated the effects of pH

on CL intensity in the range of 4.5 ~ 8.5. As can be seen in Figure S4, the probe was unperturbed under 4.5 ~ 8.5.

Considering the application of the probe in biological system, we performed CL measurements at pH 7.4 in the

range of physiological pH.

Figure S4 Effects of pH on CL intensities of PCLA-O2•−

(0.45 mg/ml) to O2•−

(0.10 µM).

S14

9. Effects of mice serum on CL response of PCLA-O2•−

to O2•−

To apply PCLA-O2•−

in more complicated biological systems, we also tested the effects of mice serum on the CL

responses of PCLA-O2•−

to O2•−

. Mice blood obtained by serial phlebotomy via the orbital sinus was centrifuged to

acquire the serum supernatant. Figure S5 indicated that PCLA-O2•−

is stable and reliably response to O2•−

in

presence of different portions of mice serum (0%-90%).

Figure S5 Effects of mice serum on CL responses of PCLA-O2•−

(0.45 mg/ml) to O2•−

(0.09 µM). CL and CL0

represent the emission intensities of PCLA-O2•−

in the presence and absence of O2•−

, respectively.

10. The stability of PCLA-O2•−

over time

With the aim to examine the stability of PCLA-O2•−

, we tested CL intensities of PCLA-O2•−

without O2•−

over a

period of 22 min. It can be found the signals of PCLA-O2•−

remained unchanged, which also revealing excellent

stability of the PCLA-O2•−

during 22 min. What’s more, upon addition of 0.10 µM O2•−

, the CL signal of

PCLA-O2•−

was increased dramatically.

S15

Figure S6 The CL intensities (black dots) output of PCLA-O2•−

(0.45 mg/ml) in the absence of O2•−

within 22 min.

CL intensity (red dot) represented that the CL signal of PCLA-O2•−

increased after reaction with O2•−

(0.10 µM).

11. CL responses of PCLA-O2•−

in MCF-10A, 4T1 extracts and macrophages extracts

Figure S7 The CL (λem=570±10 nm) changes of PCLA-O2•−

in MCF-10A, 4T1 extracts and macrophages extracts

experiments. ([PCLA-O2•−

]: 0.45 mg/ml, [SOD]: 200 U, [Tiron]: 10 µM). Inset, representative CL images of

PCLA-O2•−

in response to O2•−

. (n = 3)

12. Cytotoxicity assay and in vivo toxicity study [5]

To evaluate the toxicity of PCLA-O2•−

in 10% THF, we performed the cytotoxicity and in vivo toxicity

experiments, respectively. For cytotoxicity study, we performed MTT assay in macrophages with different probe

concentrations from 0.45 mg/mL to 2.25 mg/mL containing 10% THF, indicating PCLA-O2•−

and 10% THF low

cytotoxicity. Furthermore, we used the clinical symptoms including the weight, hair loss, scab, vomiting and

S16

diarrhea to investigate the potential toxicity of PCLA-O2•−

in KM mice after i.p. injection of PCLA-O2•−

with

experimental concentration 0.068 mg and even higher concentration 0.136 mg. The experimental data showed the

body weights of treated groups were identical to control group and no mice exhibited any symptom of hair loss,

scab, diarrhea, or vomiting over a period of 2 weeks. In the same time, the calculated dose of THF injected into KM

mice is 382 mg/kg, which is much lower than the median lethal dose (LD50) of THF, 1900 mg/kg showed in

toxicology data network.[6]

Therefore, we believe that PCLA-O2•−

and THF used in the experiments had low

toxicity to live cells and small animals.

Figure S8 a) Viability of macrophages in the presence of different concentration PCLA-O2•−

. b) Change in body

mass of mice injected with PCLA-O2•−

compared with 0.9% NaCl aqueous solution control group.

13. The CL images of endogenous O2•−

in the LPS-induced inflammatory mice over time

Figure S9 The CL (λem=570±10 nm) imaging of endogenous O2•−

in the LPS-treated mice with PCLA-O2•−

(0.068

S17

mg) were acquired using an IVIS Lumina II in vivo imaging system. Images were 0.5 min, 5 min, 10 min, 15 min,

20 min, 25 min, respectively. (I) LPS was injected into abdomen, followed by injection of PCLA-O2•−

(0.068 mg) 4

h later. (II) LPS was injected into abdomen, followed by injection Tiron (400 µL, 10 µM) 3 h later. 1 h later,

PCLA-O2•−

(0.068 mg) was injected. (III) Saline and PCLA-O2•−

(0.068 mg). (IV) The control: only saline. (n=4)

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