Supporting Information for

Neutralizing tumor-promoting inflammation with polypeptide-

dexamethasone conjugate for microenvironment modulation and

colorectal cancer therapy

Sheng Ma a,b,c, Wantong Song a,c*, Yudi Xu a,c,d, Xinghui Si a,b,c, Dawei Zhang a,c, Shixian Lv e,

Chenguang Yang a,c, Lili Ma a,c, Zhaohui Tang a,b,c, Xuesi Chen a,b,c*

a Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Changchun 130022, P. R. China

b University of Science and Technology of China, Hefei 230026, P. R. China

c Jilin Biomedical Polymers Engineering Laboratory, Changchun, 130022, P. R. China

d University of Chinese Academy of Sciences, Beijing 100039, P. R. China

e Department of Bioengineering, Molecular Engineering and Sciences Institute, University of

Washington, Seattle, 98195, USA

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Synthesis of mPEG-b-PLL block copolymer

Methoxyl poly(ethylene glycol)-b-poly(N(ε)-benzyloxycarbonyl-L-lysine) (mPEG-b-PZLL)

was synthesized by the ring-opening polymerization of Lys-NCA using mPEG-NH2 as a

macroinitiator. Typically, mPEG-NH2 (5.5 g, 1.1 mmol) were dissolved in 50.0 mL of dried DMF

in a flame-dry flask after an azeotropic dehydration process with toluene. Then ZLL-NCA

(5.0g,16.7 mmol) were dissolved in dried DMF and added to the solution of mPEG-NH 2. The

polymerization was carried out at room temperature for 7 days. Then, the solution was precipitated

into 10 times cold diethyl ether. The obtained precipitate was dried under vacuum to remove

diethyl ether (yield: 91.3%). The degree of polymerization (DP) of PZLL was determined to be 14

from proton nuclear magnetic resonance pectra (Fig. S1A).

mPEG-b-PLL was prepared by removing the protecting group of mPEG-b-PZLL. Briefly, 4.0

g of mPEG-b-PZLL was dissolved in 40.0 mL of trifluoroacetic acid. Then, 12.0 mL of hydrogen

bromide/acetic acid solution (33 wt.%) was added. After stirring for 4 h at room temperature, the

solution was precipitated into excess cold diethyl ether. The obtained product was dissolved in

water, dialyzed (molecular weight cut-off = 3500 Da) against water for 48h, and then lyophilized

with a yield of 82.3%,1H NMR of mPEG-b-P(LL-DTPA) was measured using D2O as a solvent

(Fig. S1B).

Synthesis of mPEG-b-P(LL-DTPA) and mPEG-b-P(LL-SA).

mPEG-b-PLL (1.0 g, 0.13 mmol) and DTPAA (0.76 g, 6.5 mmol) were dissolved in dry DMF

(20 mL), then triethylamine (TEA) (0.66 g, 6.5 mmol) was added into the solution. The reaction

mixture was stirring at 35 °C for 6 h. The mPEG-b-P(LL-DTPA) crude product was obtained by

repeating precipitation into excess cold diethyl ether. The crude product was dissolved in DMF

and (MWCO 3500 Da) against DMF for 24h, following dialyzing against deionized water for 48h.

The mPEG-b-P(LL-DTPA) was obtained after lyophilization. 1H NMR of mPEG-b-P(LL-DTPA)

was measured using DMSO-d6 as a solvent (Fig. S1C).

As for mPEG-b-P(LL-SA), mPEG-b-PLL (1.0 g, 0.13 mmol) and succinic anhydride (SA,

0.84 g, 8.4 mmol) were dissolved in dry DMF (20 mL). Then triethylamine (TEA) (0.68 g, 6.75

mmol) was added into the solution and the mixture. The reaction mixture was stirring at 35 °C for

6 h. The mPEG-b-P(LL-SA) crude product was obtained by repeating precipitation into excess

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cold diethyl ether. The crude product was dissolved in DMF and dialyzed against DMF (MWCO =

3500 Da) for 24 h, following dialyzing against deionized water for 48 h. The mPEG-b-P(LL-SA)

was obtained after lyophilization. 1H NMR of mPEG-b-P(LL-SA) was measured using DMSO-d6

as a solvent (Fig. S1D)

Fig. S1. 1H NMR spectra of mPEG-b-P(ZLL) in TFA-d (A), mPEG-b-PLL in D2O (B), mPEG-b-

P(LL-DTPA) (C) and mPEG-b-P(LL-SA) (D) in DMSO-d6.

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Fig. S2. 1H NMR spectra of mPEG-b-P(LL-SA) (a), DEX (b) and L-SA-DEX (c) in DMSO- d6.

Synthesis of FITC-labeled L-SS-DEX and L-SA-DEX

FITC-labeled L-SS-DEX and L-SA-DEX were prepared in a similar way. Take FITC-labeled L-

SS-DEX as an example. To a flame-dry flask, L-SS-DEX (50.0 mg) was dispersed in DMF and

FITC (5.0 mg) was added under stirring. The reaction was continued in the dark for 12 h. After

reaction, the reaction solution was immediately dialyzed against distilled water for 72h in dark. A

yellow powder was obtained as FITC-labeled L-SS-DEX after lyophilization.

Solution behavior of polypeptide-DEX conjugates

The nano-micelles of L-SA-DEX and L-SS-DEX was formed by directly dispersed in phosphate

buffered saline (PBS). Dynamic light scattering (DLS) was used to characterize the sizes of the

self-assemble nano-micelles. To estimate the stability of L-SA-DEX and L-SS-DEX in vitro, L-

SA-DEX and L-SS-DEX were dissolved in PBS containing 10% FBS, and then the solutions were

placed in a thermotank at 37 °C under gently shaking at 90 rpm. At designed time intervals (6, 12,

24, 48, and 72 h), a small volume of the solution was taken out and the size was tested with DLS.

To obtain transmission electron microscopy (TEM) images, the aqueous solution of L-SA-DEX

and L-SS-DEX was dropped onto the copper net, and then left alone until the water evaporated

completely. The TEM measurement was performed on a JEOL-JEM-1011 transmission electron

microscope (Tokyo, Japan) with an accelerating voltage of 100 kV.

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Fig. S3. Characterizations of L-SA-DEX and L-SS-DEX. (A) Hydrodynamic radii (Rh) of L-

SA-DEX and L-SS-DEX. (B) Stability test of nano-micelles formed by L-SA-DEX and L-SS-

DEX in PBS containing 10% FBS at 37°C (Data were presented as the mean ± standard deviation

(n = 3)). Typical morphologies of L-SA-DEX (C) and L-SS-DEX (D) measured by TEM.

HPLC-MS Analysis

HPLC-MS (Quattro Premier XE, USA) with a reverse-phase C-18 column (ACQUITY UPLC®

BEH) was utilized to detect the release solution obtained from the pH 7.4 with 10 mM GSH and

pH 5.5 groups at the end of the released experiment (120 h). Two mobile phases were used, with

mobile phase A consisted of 0.01% acetic acid in water, and mobile phase B of acetonitrile. A

gradient elution procedure was performed (0–1 min: 60% A, 1–2 min: linear gradient to 30% A,

2–3 min: 30% A, 3–3.2 min: linear gradient to 60% A, 3.2–6 min: 60% A) at a flow-rate of 300

μL/min. The temperatures of the column oven and autosampler tray were set at 45 oC and 5 oC,

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respectively.

As shown in Fig. S4, the ions with molecular weight of 373.33 and 355.30 were found to be

related with DEX ions (Mw =393.31), and the ions with molecular weight of 373.33 showed the

strongest abundance. To obtain a high analytical sensitivity, the instrument was operated in

“multiple reaction and monitoring” (MRM) mode, and confirmed the ions with molecular weight

of 373.33 were the daughter ions of DEX ions. Therefore, the most intense product ions with

molecular weight from 373.33 to 393.31 was used for qualitative and quantitative study. Under

this examination condition, if the retention time of the converted DEX is consistent with the

parent dexamethasone, thus the transformed DEX matches the parent compound.

Fig. S4. MS spectrum obtained by direct injection of DEX.

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Fig. S5. HPLC-MS analysis of DEX and released DEX from L-SS-DEX in condition of pH

7.4 with10 mm GSH and pH 5.5.

Table S1. The HPLC-MS result of DEX and the released medium obtained from pH7.4 plus

10 mM GSH and pH 5.5 in vitro release experiment groups.

Name Retention time Area Concentration

DEX 3.25 2357.77 100.00 ng/mL

pH 7.4 + 10 mM GSH 3.25 1669.96 70.83 ng/mL

pH 5.5 3.26 1332.38 56.51 ng/mL

Hematological analysis

Hematological analysis was carried out to study acute side effects and toxicities caused by

different DEX formulations treatments. The female BALB/c mice (6–8 weeks old) assigned into 4

groups at random (n = 5) and received the treatment of PBS, DEX (10 mg/kg, dissolved in the

8:1:1 mixture of PBS and ethanol, Cremophor EL®), L-SA-DEX (10 mg/kg, DEX) or L-SS-DEX

(10 mg/kg, DEX). The treatment schedule was the same as the in vivo antitumor experiment. The

mice were sacrificed 24 h post the last injection, both whole blood and serum were collected. The

hematological parameters were analyzed including white blood cell (WBC), red blood cells

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(RBC), hemoglobin (HGB), hematocrit (HCT), alanine transaminase (ALT), aspartate

aminotransferase (AST) and creatinine (CREA).

Fig. S6. Hematological parameters of healthy mice treated with different formulations (n =

5).

Fig. S7. Blood chemistry analysis of healthy mice treated with different formulations (n = 3).

Fig. S8. TUNEL staining of tumor sections after various treatments.

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Fig. S9. α-SMA staining of tumor sections after various treatments.

Fig. S10. COX-2 staining of CT26 tumor tissues after various treatments.

Western Blot

For in vivo western blot analysis, the harvested tumor tissues were homogenized using

radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail mix (0.1%

v/v). The cell lysates were centrifuged, and the supernatant proteins were collected. Total protein

concentration in the lysate was determined with a bicinchoninic acid protein assay kit (BCA

Protein Assay Kit, Pierce). Protein (25 μg) was mixed with 5x loading buffer and boiled for 5 min.

Protein samples were loaded into 12% SDS-PAGE gel and transferred onto a PVDF membrane.

The membrane was blocked with 5% skimmed milk for 1 h at room temperature, and was then

incubated waveringly with primary antibodies overnight at 4 oC. After three washes with PBS-

Tween 20 (PBST), the bands were subsequently incubated with horseradish peroxidase (HPR)-

conjugated goat anti-rabbit IgG secondary antibody at room temperature for 1h. After three

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washes with PBST, the bands were placed in the chemiluminescent working solution and

visualized using an enhanced chemiluminescence Western blot detection system (GE AI600). The

semi-quantitative analysis of COX-2 expression by western blot was quantified using Image J.

Fig. S11. Western blot analysisof the COX-2 expression in CT26 tumor tissues after various treatments. (A) Western blot bands of COX-2 and GAPDH in CT26 tumors after various treatments. (B) Relative COX-2 expressions in tumors of different groups compared to PBS group; n = 3.

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Fig. S12. Gating strategies and representative flow cytometric analysis images of flow cytometry analysis. (A) Gating strategies for CD8+ T cells (CD3+CD8+ population). (B) Gating strategies of Tregs (CD4+CD25+FoxP3+ population). (C) Gating strategies of M2 and M1 macrophages (M2: F4/80+CD206+ population; M1: F4/80+CD206- population). (D) Gating strategies of MDSCs (CD11b+Gr-1+ population).

Fig. S13. Flow cytometry analysis CD3+ T cells, CD4+ T cells and macrophages in tumors of

mice after various treatments, n = 4.

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Fig. S14. CD3+CD4+ immunofluorescence staining of CT26 tumor tissues after various

treatments.

Fig. S15. CD3+CD8+ immunofluorescence staining of CT26 tumor tissues after various

treatments.

Table S2. Antibodies used in the study.

Antibodies Company Catalog Application

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FITC anti-mouse CD3 Antibody BioLegend 100203 flow cyt and IFPE/Cy7 anti-mouse CD4 Antibody BioLegend 100422 flow cytAPC anti-mouse CD8a Antibody BioLegend 100712 flow cyt

APC/Cy7 anti-mouse CD25 Antibody BioLegend 102026 flow cytAPC anti-mouse CD11b Antibody BioLegend 101212 flow cytFITC anti-mouse CD206 Antibody BioLegend 141704 flow cyt

PerCP/Cy5.5 anti-mouse F4/80 Antibody BioLegend 123128 flow cytAPC/Cy7 anti-mouse I-A/I-E (MHCII)

AntibodyBioLegend 107628 flow cyt

PE anti-mouse Gr-1 Antibody BioLegend 108408 flow cytPE anti-mouse FoxP3 Antibody BioLegend 126404 flow cyt

Anti-α-SMA Antibody Abcam ab7817 IFCox-2 Antibody Abcam ab15191 IF and WB

GAPDH Antibody ABclonal AC002 WBFITC Goat anti-Rabbit IgG ABclonal AS011 IFCy5® Goat anti-Rabbit IgG ABcaml Ab6564 IFHPR Goat anti-Rabbit IgG ABclonal AS014 WB

Alexa Fluor® 594 anti-mouse CD4 Antibody Biolegend 100446 flow cyt and IFCD8a Monoclonal Antibody eBioscienc

e14-0808-82 flow cyt and IF

Table S3. ELISA kits used in the studyELISA kits Company Catalog

MOUSE-IFNgamma ELISA kit Anoric TAE-366mMouse-TNFalpha ELISA kit Anoric TAE-569m

Mouse-IL-4 ELISA kit Anoric TAE-384mMouse-IL-10 ELISA kit Anoric TAE-371m

MOUSE-PGE2 ELISA Kit Anoric TAE-497m

Table S4. Drug accumulation ratios of DEX and L-SS-DEX in tumors (CT26) to kidney, liver, lung, spleen and heart at 6, 24 and 48 h after tail vein injection.

6 h 24 h 48 hDEX L-SS-DEX DEX L-SS-DEX DEX L-SS-DEX

Tumor/Kidney 0.33±0.03 0.83±0.18 0.34±0.06 1.84±0.31 0.27±0.41 1.70±0.37Tumor/Liver 0.24±0.01 0.36±0.03 0.35±0.01 0.70±0.02 0.26±0.03 1.03±0.24Tumor/Lung 1.63±0.14 2.05±0.33 0.79±0.06 2.29±0.12 0.91±0.17 5.16±0.56

Tumor/Spleen 0.95±0.08 1.85±0.18 1.64±0.09 2.16±0.19 # 1.55±0.14Tumor/Heart # 2.28±0.15 # 4.61±0.34 # 5.93±0.48

# indicates the value is below test limit.

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