Confocal dishes

BUC cells were grown on confocal dishes (Corning Inc), rinsed twice with PBS and then fixed for 15 min using 4% paraformaldehyde in PBS on ice. Cells were washed 3 times with PBS, and then permeabilized using 0.1% Triton X‐100 in PBS on ice for 10 min. After washing twice with PBS, they were blocked using 5% BSA in PBST at 37°C for 30 min. Cells were then incubated with primary antibodies at 37°C for 1 h. Cells were washed with PBST, then cells were incubated with the corresponding secondary antibodies for 30 min at 37°C. The cells were then stained with DAPI to stain the nuclei. Fluorescent images were acquired using an OLYMPUS FV1000 confocal microscope (Olympus). Relative mean fluorescence densities were analyzed using ImageJ software (NIH) and plots were constructed using GraphPad Prism 6 software (GraphPad Software, Inc).

VSMCs were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Honokiol was purchased from Selleck Chemicals (Houston, USA, catalog no. S2310), MSNPs (catalog no. 643645) and F‑127 pluronic gel were purchased from Sigma‑Aldrich Co. (St Louis, MO, USA). Transmission electron microscope was acquired from FEI (Hillsboro, Oregon, USA). FBS and 0.25% (w/v) trypsin−EDTA were obtained from Corning (New York, USA). DMEM and penicillin−streptomycin solution were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Culture flasks (25 cm² surface area), culture plates, a filter of 8 μm pore size and confocal dishes were acquired from Corning Incorporated (Corning, NY, USA). 2F Fogarty embolectomy catheters were obtained from Edwards Lifesciences (Irvine, CA, USA). WST-1 was purchased from Beyotime Biotechnology (Shanghai, China). Foxp3/Transcription Factor Staining Buffer Set was purchased from Thermo Fisher Scientific. The primary antibodies used in Western blotting analysis were: anti-p-smad3, anti-p-ERK1/2 and anti-PCNA (Immunoway Biotechnology, NY, USA). α‑SMA antibody was obtained from Abcam (Cambridge, UK).

The amount of ∼1 × 10⁶ HepG2 cells per dish were seeded in confocal dishes (Corning, New York, NY, USA) and cultivated in a cell culture medium for 24 h. Afterwards, cells were incubated with Cy5-modified ^HPMPC@DOX or ^RPMPC@DOX at equal amounts of DOX for 1, 2, and 4 h in hypoxic environment. Hypoxia environment or normoxic environment were generated in the cells by cultivating in Biospherix C21 hypoxic chamber supplemented with 1% of oxygen or air, respectively. Cells were rinsed and stained with DAPI and observed with a Nikon laser confocal microscope (C2plus, Nikon, Tokyo, Japan). The cellular uptake of ^HPMPC@DOX or ^RPMPC@DOX was performed with flow cytometry (Olympus IX71, Tokyo, Japan). HepG2 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells per well and incubated for 24 h. Then, cells were cultivated with ^HPMPC@DOX or ^RPMPC@DOX at equal amounts of DOX for 4 h in hypoxic environment. After that, cells were washed several times with PBS and analyzed by flow cytometry (Olympus IX71, Tokyo, Japan).

To evaluate the cellular uptake of LDNLC in an MCF-7 ADR cell line (Cell Bank of Shanghai Institute of Biochemistry and Cell Biology; Chinese Academy of Sciences, Shanghai, China), MCF-7 ADR cells were seeded in confocal dishes (Φ=15 mm; Corning Incorporated, Corning, NY, USA) at a density of 1 × 10⁵ cells/dish and cultured overnight (humidified atmosphere containing 5% CO₂ at 37°C). The dishes were then supplemented with 2 mL serum-free medium (DMEM containing 10% FBS; Thermo Fisher Scientific). The culture medium mentioned in the following section was DMEM (unless otherwise stated) containing DNLC or LDNLC. Additionally, cells were incubated with 60 μM of Di (an inhibitor of NQO1) solution at 37°C for 3 h prior to nanoparticles’ addition. Following 12 h of incubation with nanoparticles, cells were fixed with 4% formaldehyde for 15 min at 37°C. Cells were qualitatively assessed using a Leica confocal laser scanning microscope (LSM 710, ×600; Carl Zeiss Meditec AG, Jena, Germany).27 (link) DOX fluorescence (excitation 510 nm, emission 550 nm) was expressed as red, Hoechst 33342 (excitation 405 nm, emission 460 nm) was expressed as blue.