H2O2-responsive and plaque-penetrating nanoplatform for mTOR gene silencing with robust anti-atherosclerosis efficacy† †Electronic supplementary information (ESI) available: Detailed experimental procedures, supporting tables and figures. See DOI: 10.1039/c7sc03582a

A H2O2-responsive and plaque-penetrating S2P–CeO2–ASOs nanoplatform was developed for the effective silencing of mTOR and treatment of atherosclerosis.

Confocal fluorescence imaging studies were performed with a TCS SP8 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens (×63).
Quantitative real time (qRT)-PCR was carried out on LineGene 9620 sequence detection system (Bioer, Hangzhou, China). In vivo imaging was performed with Caliper IVIS Lumina III (Caliper Co., USA).
Preparation of CeO 2 and S2P-CeO 2 -ASOs. CeO 2 NWs were prepared using a modified hydrothermal method. 1 Briefly, 5mL of 0.05 M Ce(NO 3 ) 3 solution was added dropwise into 35 mL of 6 M NaOH solution. The mixture was aged at room temperature under continuous stirring for 30 min. The white slurry was then transferred into an autoclave and maintained at 100 °C for 24 h to get highly crystallized CeO 2 NWs with a high aspect ratio. After cooling to room temperature, the sediment was washed by deionized water and ethanol alternately for three times and then dried at 60 ºC overnight.
To prepare the S2P-CeO 2 -ASOs nanoplatform, CeO 2 NWs was first modified with Mal-PEG-NH 2 (MW 2000) by stirring the NPs (10 mg) with the Mal-PEG-NH 2 (2 mg) in ethanol solution for 4 h at room temperature. Then, PEG-capped CeO 2 NWs were pelleted down, washed, and resuspended in HEPES (pH 7.4). Next, 10 mg of as-prepared PEG-CeO 2 NWs were reacted with 1.5 mg of S2P peptides (CRTLTVRKC) in 5 mL of HEPES (pH 8.0) for 12 h at room temperature in the presence of TCEP. Excess S2P peptide were then removed by filtration (MWCO =1000 kDa). S2P-PEGylated CeO 2 NW was obtained by coupling the thiol groups of S2P peptide with the maleimide groups on PEG. Finally, loading of siRNA was achieved by mixing S2P-PEGylated CeO 2 NWs (10 mg) and ASO1 and ASO2 (10 nM) in 10 mM HEPES (pH 7.4) with 150 mM concentrations of NaCl (1 h, 37 °C, 200 rpm shaking). Free ASOs were removed by filtration (MWCO =100 kDa). The final nanoplatforms were washed and centrifuged several times with DNase/RNAase free water and diluted to a known volume, estimated for their particle size, Zeta potential and the loaded ASOs level, prior to use in different (Beijing, China). All types of cells were cultured in Dulbecco's modified Eagles medium (DMEM) with 10% fetal bovine serum and 100 U/mL of 1% antibiotics penicillin/streptomycin and maintained at 37 °C in a 5% CO 2 /95% air humidified incubator (MCO-15AC, SANYO). To ensure maintenance of the contractile phenotype, we used cells in the second to eighth passages for each experiment. Cellular Uptake. VSMCs were seeded in 6-well plates at 5 × 10 4 cells/well 24 h prior to addition of the test substance. Then, sterilized S2P-CeO 2 -ASOs or CeO 2 -ASOs was cultured with VSMCs at a concentration of (50 μg/mL). In blocking group, excess amount of S2P peptide (1 mg/mL) was added to cells 30 min before S2P-CeO 2 -ASOs. S-5 For quantification of nanoparticles uptaken in VSMCs, ICP-AES measurements were performed. After 4 h incubation, the media was aspirated off, cells were washed twice with PBS and digested with 70% w/v nitric acid containing 10 ppm Yttrium at 80 ºC for 6 h. After cooling to room temperature, condensates were collected by centrifugation, and diluted to 5 mL with water. Samples were then filtered through a 0.45 μm hydrophilic PVDF membrane into 15 mL Falcon tubes. Standards were prepared by diluting 1000 ppm cerium standards solution to 100 ppm, 10 ppm, 1 ppm, 0.2 ppm, 0.04 ppm with 15% w/v nitric acid. ICP-AES was performed on the samples using the Optima 7300 (Perkin Elmer). Another batch of VSMCs was exposed to different concentrations (0-60 μg/mL) of S2P-CeO 2 -ASOs, and the internalization of NPs were monitored at specific time intervals. Two control cells, HUVEC 2 and HEK293, 3 were also introduced to better demonstrated the stabilin-2 specificity of S2P-CeO 2 -ASOs.
Endosomal escape. VSMCs were seeded at an initial density of 5 × 10 4 cells/dish in 6well plates or 20-mm glass bottom dishes and incubated for 24 h before adding the test substance. The endosomal escape of S2P-CeO 2 -ASOs was visualized by transmission electron microscopy (TEM) and confocal microscopy. (1) TEM measurements. Fresh medium containing sterilized S2P-CeO 2 -ASOs (50 µg/mL) were added, and the cells were incubated for 2 h and 6 h at 37 °C, respectively. After that, cells were trypsinized, centrifuged, and fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.4，0.1 M) for 1 h at room temperature and rinsed. Cells were then post fixed 1 h in 2% osmium tetroxide with 3% potassium ferrocyanide and rinsed, next enbloc staining with a 2% aqueous uranyl acetate solution and dehydration through a graded series of alcohol (50%, 70%, 80%, 95%, 100%). Then they were put into two changes of propylene oxide, a series of propylene/epon dilutions and embedded in 100% Epon. The 70 nm thin sections were cut on a Leica UC6 ultramicrotome, and images were taken on a JEOL 1200 EX (JEOL, Ltd. Tokyo, Japan) using an AMT 2k digital Camera. (2) Confocal imaging.
Fresh medium containing sterilized Cy5-ASOs loaded S2P-CeO 2 (50 µg/mL) were added, S-6 and the cells were incubated for 2 h and 6 h at 37 °C, respectively. Catalase (100 μM) was used as a H 2 O 2 scavenger. After removing the medium and subsequently washing with PBS buffer, the endosomes and nuclei were stained with Lysotracker green and DAPI, respectively. The cells were then viewed under TCS SP8 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with 405 nm, 488 nm and 563 nm excitation.
Cytotoxicity assay. VSMCs were seeded in 96-well plates (10 4 cells/well) and cultured overnight. Then fresh medium containing sterilized S2P-CeO 2 -ASOs (50 μg/mL) was added to each well and incubated for 2, 6, 12 and 24 h, respectively. Cell viability was evaluated by MTT assay according to the previously reported method. 4 Cells incubated with free medium served as a negative control.
In vitro silencing efficacy. VSMCs were seeded in 6-well cell plates or glass slides  (Table S3). The expression of mTOR was analyzed using GAPDH as the internal reference. (2) Western blotting. Each group of cells was lysed in RIPA buffer supplemented with cocktail protease inhibitors and PMSF (1 mM), respectively. The amount of protein was measured using a protein assay kit (Beyotime, China). Equal amounts of protein (50 μg) were resolved on 6% SDS-PAGE gels and S-7 electro-blotted onto a polyvinylidene fluoride (PVDF) membrane and blocked with TBS containing 0.05% Tween-20 and 5% nonfat milk powder for 1 h. Next, membranes were incubated overnight with primary antibodies against mTOR (1:1,000) and β-actin Pharmacokinetics study. Healthy ApoE -/mice were randomly divided into three groups (n = 3) and given an intravenous injection of (i) free Cy5-labelled ASOs, (ii) Cy5labelled CeO 2 -ASOs, (iii) Cy5-labelled S2P-CeO 2 -ASOs at 0.5 mg/kg ASOs dose. At predetermined time intervals, 20 µL of tail vein blood was withdrawn using a tube S-9 containing heparin, and the wound was pressed for several seconds to stop the bleeding.
For group (ii) and (iii), 1mM H 2 O 2 was added to release the loaded Cy5-labelled ASOs.
Then, fluorescence intensity of Cy5-labelled ASOs in the blood was determined by an RT 6000 microplate reader. The blood circulation half-life (t 1/2 ) was calculated from a firstorder fit of the data.
Biodistribution study. Plaque-bearing ApoE -/mice were randomly divided into four groups (n = 3) and given an intravenous injection of (i) PBS, (ii) free Cy5-labelled ASOs, In vivo silencing efficacy. ApoE -/mice (6-8 weeks) fed with the high-fat diet were randomly divided into four groups (n = 3) and given an intravenous injection of (i) PBS, (ii) free ASOs, (iii) CeO 2 -ASOs, (iv) S2P-CeO 2 -ASOs at 0.5 mg/kg ASOs dose. After three days post-injection, aortas of each group were harvested. To evaluate in vivo silencing efficacy, aortas were homogenized on ice in lysis buffer, respectively. Changes in mTOR expression and LC3II/LC3I ratio were analyzed by western blot following the S-10 same procedure as described above (primary antibody against mTOR: 1:1,000 and LC3B: 1:1,000).
The (v) group of mice was each injected with 4 mg/kg 6  Images were captured using a Leica DMI3000 microscope (Leica Microsystems, Wetzlar, GmbH). The body weight of each mouse were monitored and recorded twice a week.

Statistical analysis.
Each experiment was repeated three times in duplicate if not stated otherwise. Data were presented as mean ± S.D. Comparisons between groups were analyzed using Student's t-test, P < 0.05 was considered statistically significant. Table S1: Sequences of antisense oligonucleotides with modifications used for this study.

Fig. S17
Representative histology (H&E) images of major organs collected from high-fat diet-fed ApoE -/mice receiving treatments as indicated after 12 weeks. No noticeable abnormality was found in the heart, liver, spleen, lung, or kidney. Scale bar = 100 μm.