Lysotracker

To further analyze the subcellular localization of sericin-PBLG-DOX after cellular uptake, ADR cells were seeded in CLSM dishes overnight, pre-incubated with 250 nM Lysotracker (KeyGEN, Nanjing, China) for 30 min at 37 °C, and then incubated with DOX or sericin-PBLG-DOX for a further 12 h. Finally, the cells were washed, fixed with 4% paraformaldehyde, stained with DAPI, and imaged by CLSM.
To determine whether the nanomicelles release DOX caused by low pH of lysosome, the seeded cells were pre-incubated with NH₄Cl (20 nM) for 30 min. Then the cells were treated with sericin-PBLG-DOX and further observation by CLSM.

Ferric acetylacetonate (AR, Sinopharm Chemical Reagent Co. Ltd., SCRC, Shanghai, China), oleic acid (85%, Aladdin, Shanghai, China), benzyl ether (98%, Sigma-Aldrich, St. Louis, MS, USA), ethanol (AR, SCRC), n-hexane (97%, SCRC), dimercaptosuccinic acid (98%, TCI, Shanghai, China), acetone (AR, SCRC), triethylamine (AR, Aladdin, Shanghai, China), methanol (AR, SCRC), polyethyleneimine (Mw 25000, Sigma-Aldrich, St. Louis, MS, USA), heptafluorobutyric anhydride (97%, Macklin, Shanghai, China), amino-polyethylene glycol-carboxyl (Mw 2000, Xi’an Ruixi, Xi’an, China), methoxy-polyethylene glycol-polyethyleneimine (mPEG-PEI, Mw 2000-25000, Xi’an Ruixi Co. Ltd., Xi’an, China) FITC (Beyotime, Shanghai, China), CCK8 kit (KeyGen, Nanjing, China), Hoechst33342 (KeyGen, Nanjing, China), rhodamine-phalloidin (KeyGen, Nanjing, China), LysoTracker (KeyGen, Nanjing, China), Annexin V-FITC/PI kit (KeyGen, Nanjing, China), Anti-CXCR4 antibody (Abcam of Thermo Fisher Scientific Inc., Waltham, MA, USA), DMEM high glucose medium (KeyGen, Nanjing, China), fetal bovine serum (Gibco of Thermo Fisher Scientific Inc., Waltham, MA, USA), trypsin (Gibco of Thermo Fisher Scientific Inc., Waltham, MA, USA), siRNA(GenePharma Co. Ltd., Shanghai, China), 4 T1, A549, HeLa cell line was provided by Southeast University School of Medicine (Nanjing, China).

The JBMMSC were seeded on the glass slides until 80% confluence. Then Lyso Tracker (KeyGEN, Cat#KGMP006) was added and incubated with cells at 37°C for 1 hour. After that, cells were fixed in 4% paraformaldehyde. Cells were permeabilized in PBS containing 0.5% Triton X‐100 for 20 minutes and blocked with normal goat serum for 30 minutes at room temperature. Then, the cells were incubated overnight at 4°C with a rabbit anti‐CTSK polyclonal antibody (Abcam, Cat#ab19027). After that, the sections were incubated with goat anti‐rabbit IgG (H + L) (Proteintech, Cat#SA0013‐2) at 37°C for 30 minutes. Nuclei were counterstained with DAPI.