Supporting Information

of

Redox-responsive polyprodrug nanoparticles for targeted siRNA

delivery and synergistic liver cancer therapy

Senlin Li,[a,b,c] Phei Er Saw,[a,b] Chunhao Lin,[a,b] Yan Nie,[a,b] Wei Tao,[d] Omid C.

Farokhzad,*[d] Lei Zhang,[a,b,c]and Xiaoding Xu*[a,b]

[a] Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene

Regulation, Medical Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen

University, Guangzhou 510120, P. R. China

[b] RNA Biomedical Institute, Sun Yat-sen Memorial Hospital, Sun Yat-sen University,

Guangzhou 510120, P. R. China

[c] Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen

University, Guangzhou, China

[d] Center for Nanomedicine and Department of Anesthesiology, Brigham and Women’s

Hospital, Harvard Medical School, Boston, MA 02115, USA

* Corresponding authors: ofarokhzad@bwh.harvard.edu; zhangl9@mail.sysu.edu.cn;

xuxiaod5@mail.sysu.edu.cn

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1. Synthesis of LA conjugated DSPE-PEG3k (DSPE-PEG3k-LA)

DSPE-PEG3k-NH2 (102mg, 0.03 mmol), LA (107 mg, 0.3 mmol), EDC.HCl (57.5 mg, 0.3

mmol), and NHS (34.5 mg, 0.3 mmol) were dissolved in deionized water and the mixture was

stirred at room temperature for 48 h. Subsequently, the mixture was transferred to dialysis

tube (MWCO 1000) and dialyzed against deionized water for 48 h. The final DSPE-PEG3k-

LA was collected as white powder after freeze-drying. The synthesis route is shown in

Scheme S1.

Scheme S1. Synthesis route of DSPE-PEG3k-LA.

2. Gel permeation chromatography (GPC)

Number- and weight-average molecular weights (Mn and Mw, respectively) of the polymers

were determined by Agilent Technologies 1260 Infinity. DMF containing 10 mM LiBr was

used as the eluent at a flow rate of 0.5 mL/min. Waters millennium module software was used

to calculate molecular weight on the basis of a universal calibration curve generated by linear

PMMA standard of narrow molecular weight distribution.

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3. High-performance liquid chromatography (HPLC)

HPLC analysis was performed on a Waters 2690D HPLC system, equipped with a Waters

model 510 HPLC pump, a UV-Vis detector and a Symmetry C18 column. The HPLC analysis

was conducted by using a linear gradient of methanol and DI water at a flow rate of 0.5

mL/min. The detector was set at 365 nm for data collection and analysis.

4. Evaluation of redox response of the polyHCPT

GPC and HPLC analysis were used to evaluate the redox response of the polyprodrug. The

polyprodrug (2 mg) was dissolved in 2 mL of DMF/H2O (9:1, V/V) and then GSH (6.2 mg,

0.02 mmol) was added to obtain a solution with GSH concentration of 10 mM.At

predetermined intervals, 100 μL of the solution was taken for GPC and HPLC analysis. For

the mixed solvent of DMF and H2O, it can well dissolve the hydrophobic polyHCPT to

facilitate the redox response of the polyprodrug.

5. Preparation of FRET pair loaded nanoparticles (NPs)

The polyHCPT and DSPE-PEG3K were respectively dissolved in DMF to form a homogenous

solution with a concentration of 5 mg/mL. Subsequently, 10 μL of DMF mixture of FRET

pair of coumarin 6 and Nile red (1:10 in molar ratio) was prepared and mixed with 100 μL of

polyHCPT solution, 80 μL of DSPE-PEG3K solution, 1 nmol siLuc (0.1 nmol/µL aqueous

solution) and 20 μL of G0-C14 solution (5 mg/mL in DMF). Under vigorous stirring (1000

rpm), the mixture was added dropwise to 5 mL of deionized water. The NP dispersion formed

was transferred to an ultrafiltration device (EMD Millipore, MWCO 100 K) and centrifuged

to remove the organic solvent and free compounds. After washing with PBS solution (pH 7.4)

(3 × 5 mL), the FRET pair loaded NPs were dispersed in the PBS solution at a siRNA

concentration of 1 nmol/mL.

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6. Evaluation of intracellular redox response of the NPs

Luc-HeLa (50,000 cells) were seeded in round discs and incubated in 2 mL of RPMI1640

medium containing 10% FBS for 24 h. After replacing the medium with 2 mL of fresh

medium, NPs loaded with FRET pair of coumarin 6 and Nile red were added, and the cells

were allowed to incubate for different time. After removing the medium and subsequently

washing with PBS buffer thrice, the cells were fixed with 4% paraformaldehyde and then

views under CLSM under the excitation of 400 nm and emission of 510 and 590 nm.

7. Western Blot

Equal amounts of protein, as determined with a bicinchoninic acid (BCA) protein assay kit

(Pierce/Thermo Scientific) according to the manufacturer’s instructions, were added to SDS-

PAGE gels and separated by gel electrophoresis. After transferring the proteins from gel to

polyvinylidene difluoride membrane, the blots were blocked with 3% BSA in TBST (50 mM

Tris-HCl pH 7.4, 150 mM NaCl, and 0.1% Tween 20) and then incubated in the mixture of β-

actin rabbit antibody (Cell Signaling, 13E5, 1/2500 dilution) with Bcl-2 rabbit antibody

(Abcam, ab182858, 1/2000 dilution), caspase 9 (Abcam, ab202068, 1/2000 dilution), or

topoisomerase I (Topo I, Abcam, ab109374, 1/2000 dilution). The protein expression was

detected with horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG

HRP-linked antibody, Cell Signaling, 7074, 1/1000 dilution) and an enhanced

chemiluminescence (ECL) detection system (Pierce).

8. Immune response

Healthy female BALB/c mice were randomly divided into three groups (n = 3) and given an

intravenous injection of either (i) PBS, (ii) naked siBcl-2 (0.3 mg/kg siRNA dose), (iii) siBcl-

2 loaded NP85 or (iv) LA-NP85 (0.3 mg/kg siRNA dose, 5 mg/kg HCPT-equivalent dose).

Twenty-four hours after injection, blood was collected and serum isolated for measurements 4

of representative cytokines (TNF-α, IL-6, IL-12, and IFN-γ) by enzyme-linked

immunosorbent assay or ELISA (PBL Biomedical Laboratories and BD Biosciences)

according to the manufacturer’s instructions.

9. Blood and histological analysis

Healthy female BALB/c mice were randomly divided into four groups (n = 3) and given an

intravenous injection of either (i) PBS, (ii) naked siBcl-2 (0.3 mg/kg siRNA dose), (iii) siBcl-

2 loaded NP85 or (iv) LA-NP85 (0.3 mg/kg siRNA dose, 5 mg/kg HCPT-equivalent dose).

After three consecutive injections, the blood was collected 24 h post the final injection and

serum isolated for measurements of representative blood parameters (AST, ALT, albumin,

ALKP, BUN, creatinine, and total protein). Simultaneously, the main organs were collected,

fixed with 4% PFA, and embedded in paraffin. Tissue sections were stained with

hematoxylin-eosin (H&E) and then viewed under an optical microscope.

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Fig. S1. 1H-NMR spectrum of polyHCPT in CDCl3.

Fig. S2. GPC (A) and HPLC (B) profiles of polyHCPT incubated in the mixture of DMF and

H2O (9:1. v/v) containing GSH (10 mM).

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Fig. S3. Chemical structure of G0-C14.

Fig. S4. Physiochemical characteristics of the siLuc loaded NPs (For all the NP formulations,

1 nmol siLuc was used to prepare the siRNA loaded NPs. EE% and LL% represent the

encapsulation efficiency and drug loading level, respectively)

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Fig. S5. (A, B) TEM images of the siLuc loaded NP85 incubated in the PBS solution (A) and

10 μM GSH-containing PBS solution for 24 h (B). (C) Size of the siLuc loaded NP85

incubated in the PBS containing 10% FBS for different times.

Fig. S6. Cumulative release of siRNA and HCPT from the siLuc loaded NP85 incubated in

the GSH-containing PBS solution at pH 6.0.

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Fig. S7. (A) Chemical structure of the non-redox-responsive HCPT-based polyprodrug

(denoted control polyprodrug); (B, C) TEM images of the siLuc loaded control polyprodrug

NPs incubated in the PBS solution (B) and 10 mM GSH-containing PBS solution for 24 h

(C); (D) Cumulative siRNA release profile of the siLuc loaded control polyprodrug NPs

incubated in the PBS solution containing GSH at different concentrations.

Fig. S8. Flow cytometry analysis (A) and mean fluorescence intensity (MFI, B) of L02 and

HepG2 cells treated with the FITC-labeled ASGP antibody.

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Fig. S9. CLSM images of HepG2 cells incubated with the Cy3-labeled siLuc loaded LA-

NP85 for 1 and 4 h. The endosome and nuclei were stained with Lysotracker green (green

fluorescence) and Reddot1 (blue fluorescence), respectively.

Fig. S10. Bcl-2 expression (determined by Western Blot analysis) in HepG2 cells treated with

the siBcl-2 loaded NP85 and LA-NP85. siLuc loaded NP85 and LA-NP85 were respectively

used as control.

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Fig. S11. IC50 values of siBcl-2 and HCPT in different therapeutic formulations.

Fig. S12 Western Blot analysis of Bcl-2 expression in the tumor tissues of the HepG2

xenograft tumor-bearing nude mice treated with siLuc loaded NP85 (Control NPs), and siBcl-

2 loaded NP85 and LA-NP85.

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Fig. S13. Body weight of HepG2 xenograft tumor-bearing nude mice treated with PBS, naked

siBcl-2, free HCPT, siLuc loaded NP85, and siBcl-2 loaded NP85 and LA-NP85.

Fig. S14. Histological section of the major organs of HepG2 xenograft tumor-bearing nude

mice treated with PBS, naked siBcl-2, free HCPT, siLuc loaded NP85, and siBcl-2 loaded

NP85 and LA-NP85. Hematoxylin-eosin; magnification 100 ×

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Fig. S15. Serum levels of TNF-α, IFN-γ, IL-6 and IL-12 at 24 h post injection of PBS, naked

siBcl-2, siLuc loaded NP85, and siBcl-2 loaded NP85 and LA-NP85.

Fig. S16. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT),

albumin, alkaline phosphatase (ALKP), blood urine nitrogen (BUN), creatinine, and total

protein (TP) after three consecutive injections of PBS, naked siBcl-2, siLuc loaded NP85, and

siBcl-2 loaded NP85 and LA-NP85.

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