Supporting information for 1
Investigation of the intracellular oxidative stress amplification, safety and anti-2
tumor effect of a kind of novel redox-responsive micelles 3
4
1. Experimental section 5
1.1 In vivo pharmacokinetic and biodistribution studies 6
Eighteen SD rats were randomly assigned to three groups (n = 6 per group) according 7
to the form of PTX administered (a single dose of 6 mg/kg i.v. through the tail vein): 8
free PTX, FSST-PTX or FT-PTX. At time points of 0 (pre-dose), 0.083, 0.17, 0.5, 1, 2, 9
4, 8, 12 and 24 h post injection, blood (0.3 mL) was collected into heparinized 10
polyethylene tubes via jugular veins and instantly centrifuged at approximately 4,000 11
rpm for 10 min followed by supernatant plasma (100 μL) collected and stored at -70 ºC 12
prior to analysis by HPLC. Methanol (400 μL) was added to the rat plasma samples and 13
stirred vigorously for 1 min. Then, the mixture was centrifuged at 10,000 rpm for 10 14
min. An aliquot of the upper organic phase (400 μL) was transferred to another tube 15
and evaporated to dryness at 40 ºC with nitrogen. The resulting extract was dissolved 16
in of methanol (150 μL) and mixed. After centrifugation at 13,000 rpm for 10 min, the 17
supernatant (100 μL) was taken for HPLC analysis. Winnonlin 5.2 software was utilized 18
to analyze the pharmacokinetic parameters: peak concentration (Cmax), area under the 19
plasma concentration-time curve (AUC), elimination half-life (T1/2) and mean 20
residence time (MRT) of PTX for each formulation. 21
For biodistribution studies, the xenograft MCF-7 tumor-bearing nude mice were 22
intravenously injected with free PTX, FSST-PTX or FT-PTX at PTX dose of 10 mg/kg. 23
At time points of 0.5, 2, 4, 8, 12 and 24 h post injection, blood samples were collected 24
and mice were sacrificed by cervical dislocation. Tissues were excised and lightly 25
rinsed with saline, weighed and stored at -20 ºC until assay. The concentration of PTX 26
in plasma and homogenized tissues was determined by HPLC. 27
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Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2018
2. Results 31
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2.1 Characterization of FSST and FT 33
Figure S1-S5 showed the 1H-NMR spectrum of Pluronic F127, F127-p-NPC, F127-34
APD, F127-APD-p-NPC, and F127-Cys. All the chemical shifts were expressed in parts 35
per million () relative to the solvent signal. 36
1H-NMR spectrum analysis of F127: 1H-NMR (600 MHz, CDCl3, δ): 3.42-3.51 37
(m, 4H×196, CH2CH2O of PEO), 3.32-3.33 (m, 3H×67, CH2CHO of PPO), 1.03-1.05 38
(t, 3H×67, CH3) (Figure S1). 39
1H-NMR spectrum analysis of F127-p-NPC: 1H-NMR (600 MHz, CDCl3, δ): 8.27-40
8.36 (d, 2H, Ar H), 7.39-7.51(d, 2H, Ar H), 3.39-3.64 (m, 4H×196, 3H×67, CH2CH2O 41
of PEO and CH2CHO of PPO), 1.13-1.15 (t, 3H×67, -CH3). The peaks appeared at 8.27-42
8.36 and 7.39-7.51 belonged to the benzene ring of p-NPC (Figure S2). 43
1H-NMR spectrum analysis of F127-APD: 1H-NMR (600 MHz, CDCl3, δ): 4.24 44
(s, 1H, CH), 3.75-3.77 (t, 2H, CH2), 3.71-3.73 (t, 2H, CH2), 3.39-3.66 (m, 4H×196, 45
3H×67, CH2CH2O of PEO and -CH2CHO of PPO), 1.13-1.15(t, 3H×67, CH3). New 46
peaks appeared at 4.24 and 3.75-3.77, which were referred to the ortho-dihydroxy 47
groups of APD. These peaks which belonged to p-NPC disappeared (Figure S3). 48
1H-NMR spectrum analysis of F127-APD-p-NPC: 1H-NMR (600 MHz, CDCl3, δ): 49
8.28-8.36 (d, 2H, Ar H), 7.39-7.51 (d, 2H, Ar H), 5.60 (s, 1H, CH), 4.31-4.44 (d, 2H, 50
CH2), 3.76-3.81 (d, 2H, CH2), 3.40-3.65 (m, 4H×196, 3H×67, CH2CH2O of PEO and 51
CH2CHO of PPO), 1.13-1.15 (t, 3H×67, CH3). The characteristic peaks of benzene ring 52
appeared at 8.28-8.36 and 7.39-7.51 after being activated by p-NPC (Figure S4). 53
1H-NMR spectrum analysis of F127-Cys: 1H-NMR (600 MHz, CDCl3, δ): 5.61 (s, 54
1H, CH2), 4.24 (s, 1H, CH2), 3.72-3.76 (d, 2H, CH2), 3.39-3.64 (m, 4H×196, 3H×67, 55
CH2CH2O of PEO and CH2CHO of PPO), 3.18-3.21 (d, 1H, CH2), 2.59(s, 1H, CH2), 56
1.13-1.15 (t, 3H×67, CH3). After the reaction with cystamine dihydrochloride, the 57
amide bonds were formed. Thereby, the peaks of benzene ring disappeared and new 58
peaks appeared at 3.18-3.21 and 2.59, which were attributed to the methylene protons 59
near the disulfide bonds (Figure S5). 60
1H-NMR spectrum analysis of FSST: 1H-NMR (600 MHz, CDCl3, δ): 5.60 (s, 1H, 61
CH2), 4.25-4.27 (t, 1H, CH2OH), 4.20 (s, 1H, CH2), 3.64-3.77 (m, 42H, CH2CH2O), 62
3.40-3.64 (m, 4H×196, 3H×67, CH2CH2O of PEO and CH2CHO of PPO), 3.18-3.23 (d, 63
1H, CH2), 2.91-2.94 (t, 1H, CH2), 2.78-2.80 (t, 1H, CH2), 2.62-2.66 (t, 1H, CH2), 2.58 64
(s, 1H, CH2), 1.96-2.08 (t, 3H, CH3), 1.75-1.81 (m, 1H, CH2), 1.22-1.52 (m, 10H, CH2), 65
1.13-1.15 (t, 3H×67, CH3),0.83-0.87 (m, 5H, CH3). 66
1H-NMR spectrum analysis of FT: 1H-NMR (600 MHz, CDCl3, δ): 5.60 (s, 1H, 67
CH2), 4.25-4.27 (t, 1H, CH2OH), 4.20 (s, 1H, CH2), 3.64-3.77 (m, 42H, CH2CH2O), 68
3.40-3.64 (m, 4H×196, 3H×67, CH2CH2O of PEO and CH2CHO of PPO), 2.91-2.94 (t, 69
1H, CH2), 2.78-2.80 (t, 1H, CH2), 2.83-2.71 (t, 1H, CH2), 1.96-2.08 (t, 3H, CH3), 1.75-70
1.81 (m, 1H, CH2), 1.22-1.52 (m, 10H, CH2), 1.13-1.15 (t, 3H×67, CH3), 0.83-0.87 (m, 71
5H, CH3). 72
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2.2 In vivo pharmacokinetic studies 74
The plasma PTX concentration vs. time profiles observed for PTX, FSST-PTX and FT-75
PTX were shown in Figure S7A. Two micelles achieved larger AUC and longer half-76
life compared to PTX (P < 0.01), while PTX was cleared from the circulation at 8 h 77
after administration. FSST-PTX and FT-PTX significantly prolonged the in vivo 78
circulation time of PTX. The pharmacokinetics parameters were summarized in Table 79
S4, FSST-PTX and FT-PTX extended the elimination half-life (T1/2) of P TX from 2.78 80
h (PTX) to 7.29 h and 9.01 h, respectively (P < 0.01). Meanwhile, MRT values of FSST-81
PTX and FT-PTX were 3.67-fold and 4.97-fold higher than PTX, respectively (P < 82
0.01). Moreover, the AUC0~∞ increased by about 2.58-fold for FSST-PTX and 3.44-fold 83
for FT-PTX compared to PTX (P < 0.01). 84
The in vivo biodistribution of PTX, FSST-PTX and FT-PTX after intravenous 85
administration was evaluated by quantitatively detecting the PTX concentration in 86
different tissues. Figure S7B showed the comparison of AUC0~24 among PTX, FSST-87
PTX and FT-PTX in various tissues. FSST-PTX and FT-PTX exhibited similar 88
distributions in each organ. Their distribution in tumor were 1.75-fold and 1.68-fold 89
higher than that of PTX. Moreover, their distribution in the heart, liver and spleen was 90
less than that of PTX, but higher in plasma than the latter. 91
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Figure S1 The 1H NMR image of F127 115
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Figure S2 The 1H NMR image of F127-p-NPC 120
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Figure S3 The 1H NMR image of F127-APD 126
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Figure S4 The 1H NMR image of F127-APD-p-NPC. 131
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Figure S5 The 1H NMR image of F127-APD-Cys. 138
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Figure S6 Different tissues collected after i.v. administration of different materials for 158
H&E Staining. (A) male mice. (B) female mice. 159
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Figure S7 In vivo pharmacokinetic and biodistribution studies. (A) Plasma 165
concentration-time curves of PTX, FSST-PTX and FT-PTX after i.v. administration to 166
SD rats at the PTX dose of 6 mg/kg (mean ± SD, n=6). (B) The comparison chart of 167
area under the curves (AUC0~24) of PTX in the heart, liver, spleen, lung, kidney, plasma 168
and tumor of the xenograft MCF-7 tumor-bearing nude mice following i.v. 169
administration of PTX, FSST-PTX or FT-PTX at a PTX dose of 10 mg/kg for 24 h 170
(mean ± SD, n=4). The unit of AUC0~24 in plasma is h·μg/mL. The units of AUC0~24 in 171
other organizations is h·μg/g. **P＜0.01: significantly different from PTX. 172
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Figure S8 Histological study of tissues of heart, liver, spleen, lung and kidney after 186
treatment by H&E staining (20×). 187
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Table S1 Pharmacokinetic parameters of different PTX preparations after i.v. 204
administration to SD rats at the same 6 mg/kg PTX dose (mean ± SD, n=6). **P＜0.01: 205
significantly different from PTX group. 206
Parameters PTX FSST-PTX FT-PTX
Cmax (ng/mL) 17087 ± 2433 18972 ± 1567 18669 ± 1749
T1/2 (h) 2.78 ± 0.39 7.29 ± 0.85** 9.01 ± 2.08**
AUC0~24(h·ng/mL) 13158 ± 651 33974 ± 5136** 45327 ± 5143**
MRT (h) 1.80 ± 0.16 6.61 ± 0.87** 8.95 ± 2.47**
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