Supplementary InformationDevelopment of a Multi-Target Peptide for Potentiating Chemotherapy by Modulating Tumor Microenvironment

Table S1. The primers for RT-PCR reactions [1].Primer Applied biosystems/ref (5'to3')

Mouse IL-6FORWARD: CGGAGAGGAGACTTCACAGAG

REVERSE:CATTTCCACGATTTCCCAGA

Mouse TNF-αFORWARD: TATGGCTCAGGGTCCAACTC

REVERSE:GGAAAGCCCATTTGAGTCCT

Mouse CCL2FORWARD: ATGCAGTTAACGCCCCACTC

REVERSE:CCCATTCCTTCTTGGGGTCA

Mouse IL-10FORWARD: GCCTTATCGGAAATGATCCA

REVERSE:AGGGTCTTCAGCTTCTCACC

Mouse TGF β1FORWARD: ATTCCTGGCGTTACCTTGG

REVERSE:AGCCCTGTATTCCGTCTCCT

Mouse β-actinFORWARD: CAGGTCCAGACGCAGGATGGC

REVERSE: CTACAATGAGCTGCGTGTGG

Table S2. Primary antibodies used in this studyAntigen Antibody Dilution Vendor

CD206 Rabbit-anti-CD206 Pab 1:100 Abcam, Cambridge, UKCD34 Goat-anti- CD34 Pab 1:200 R&D systems, Minneapolis, MN, USAKi-67 Rabbit-anti- Ki-67 Pab 1:100 Millipore, Darmstadt, GermanyCD4 Mouse-anti-CD4 Mab 1:50 R&D systems, Minneapolis, MN, USACD8 Mouse-anti-CD8 Mab 1:100 Abcam, Cambridge, UK

Foxp3 Mouse-anti-Foxp3 Pab 1:500 Abcam, Cambridge, UKCD11b Rabbit-anti-CD11b Pab 1:200 Novus biologicals, Colorado, USAGr-1 Mouse-anti-Gr-1 Mab 1:50 R&D systems, Minneapolis, MN, USA

CD31 Goat-anti-CD31 Pab 1:100 Origo, GermanyCD68 Mouse-anti-CD68 Mab 1:100 DAKO, DenmarkGlut-1 Rabbit-anti-Glut-1 Pab 1:400 Abcam, Cambridge, UK

CD cluster of differentiation, Mab monoclonal antibody, Pab polyclonal antibody.

Table S3. The main pharmacokinetic parameters of free Dox, PEG-Lipo-Dox, iRGD-Lipo-Dox and nRGD-Lipo-Dox in rats (n = 5).Parameters Dox PEG-Lipo-Dox iRGD-Lipo-Dox nRGD-Lipo-DoxAUC(0-t)

a (µg·mL-

1·h)11.273±2.389 1134.733±225.043 632.102±182.25*** 904.852±273.115*

AUC(0-∞)a (µg·mL-

1·h)11.667±2.389 1146.869±233.422 632.586±181.91*** 908.2±276.18*

MRT(0-t)a (h) 7.95±1.217 11.112±1.977 7.267±2.496 8.352±1.748

MRT(0-∞)a (h) 12.373±6.572 11.88±2.533 7.344±2.447 8.595±1.862

Cmaxa (µg/mL) 3.147±0.538 149.482±13.916 147.173±25.333 152.135±7.799*

T1/2za (h) ＜5 min 10.089±4.357 7.164±2.644 9.32±1.178***

CLza (mL·h-1·g) 442.469±83.595 4.502±0.884 8.444±2.384 5.918±1.709

Data represent mean ± SD (n = 5). *, p < 0. 05, ***, p < 0. 01 vs control.

Fig. S1. The chemical structure of nRGD.

Fig. S2. nRGD was cleaved to afford iRGD after treatment of legumain. Mass-Spectrometry of intact FITC-nRGD (A) and FITC-iRGD (B) and peptide fragments of nRGD after treatment of legumain (C).

Fig. S3. The average signals of Dox groups in normal organs and tumors at 48 h after single administration of Dox, PEG-Lipo-Dox, iRGD-Lipo-Dox, nRGD-Lipo-Dox (Dox equivalent, dose of 5 mg/kg) and normal saline counted using Maestro in-vivo imaging system (n = 3). *, p < 0.05, ***, p < 0.01.

Fig. S4. Cellular uptake of free Dox，PEG-Lipo-Dox，iRGD- Lipo-Dox and nRGD- Lipo-Dox in HUVEC

cells (n = 3, mean ± SD). *, p < 0.05, ***, p < 0.01.

Fig. S5. In vitro cytotoxicities of Dox groups against 4T1, HUVEC and RAW 264.7 cells at 37 °C for 24 h. The cytotoxicity of Dox groups against 4T1 (A), CoCl2 induced 4T1 (B), RAW 264.7 (C), LPS induced RAW 264.7 (D), IL-4 induced RAW 264.7 (E) and HUVEC (F). Data represent mean ± SD (n = 3). *, p < 0.05, ***, p < 0.01.

Fig. S6 Inhibiting effect of nRGD on the cell uptake of nRGD- Lipo-Dox by 4T1 and RAW 264.7 cells. Data represent mean ± SD (n = 3). *, p < 0.05, ***, p < 0.01.

Fig. S7 Histological evaluations for different organs (heart, liver, spleen, lung, kidney and bone) of tumor bearing mice on day 20 after twice administrations of Dox formulations at an equivalent dose of 5 mg/kg. Tissue sections were stained with hematoxylin and eosin and imaged at magnification of 200× except for bone at 100×. Lesions of the organs were indicated with black arrows.

References

[1] Xu Z, Wang Y, Zhang L, Huang L. Nanoparticle-delivered transforming growth factor-beta siRNA enhances vaccination against advanced melanoma by modifying tumor microenvironment. ACS nano. 2014;8:3636-45.