Lysotracker green

CLSM was also used to observe the lysosomal escape of siCy5. HaCaT cells were seeded in 35 mm dishes with glass bottoms (20,000 cells/well) for 24 h. Then, the cells were stained with LysoTracker Green (KeyGen, Nanjing, China, 1:1000) for 1 h, incubated with GT/siCy5 for 4 h, rinsed and incubated in fresh medium for another 8 h. The cells were then fixed with 4 % PFA for 15 min, counterstained with Hoechst 33342 (1 µg/ml) for 2 min and observed with the Zeiss LSM800 confocal microscope.

Methacrylic acid (MAA), divinylbenzene (DVB), 2,2-azobis (isobutyronitrile) (AIBN) were purchased from Shanghai Aladdin Chemistry Co. Ltd., acetonitrile (ACN) and methanol were purchased from Shanghai Lingfeng Chemical Reagent Co. Ltd., ethanol was purchased from Shanghai Zhenxing Chemistry Co. Ltd., iron (III) chloride hexahydrate (FeCl₃·6H2O), potassium chloride (KCl), magnesium chloride hexahydrate (MgCl₂·6H₂O), edetate disodium (EDTANa₂·2H₂O), Tris, hydrochloric acid (HCl) and sucrose were all purchased from Sinopharm Chemical Reagent Co. Ltd.. Protease inhibitor, high glucose DMEM medium, fetal bovine serum (FBS), penicillin–streptomycin, phosphate buffer saline (PBS) and trypsin enzyme were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CCK-8 kit was purchased from Dojindo Laboratories (Tokyo, Japan). DAPI, Lyso-tracker green and cell viability (staining of living and dead cells) assay kit were purchased from KeyGen Biotech (Nanjing, Jiangsu, China). Triton X-100 and γ-H2AX antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cypate was synthesized as previously described [46 (link)]. Deionized water used in all experiments was obtained from Milli-Q water system.

For confocal imaging, 4T1 cells were seeded in Lab-Tek 8-well chamber slides (1 × 10⁴ cells/well) and incubated for 1 d. Then, the cells were incubated with various drugs. Confocal images were taken by the confocal microscope.
To study the cellular internalization of DOX and HDDA, 4T1 cells were treated with DOX or HDDA (DOX: 1.8 μg/mL) for various time periods. Next, the media were replaced by the Hoechst working solution (10 μg/mL) and further incubated for 10 min. Then, the cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst) and 552 (for DOX) nm, respectively.
To study the endo-lysosomal escape of HDDA NBs, 4T1 cells were incubated with HDDA NBs (DOX: 3.6 μg/mL) for different time periods. Next, the culture media were replaced by the media containing Hoechst (10 μg/mL) and LysoTracker Green (0.5 μg/mL, purchased from KeyGEN BioTECH, China) and further incubated for 15 min. Finally, the 4T1 cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst), 488 (for LysoTracker Green), and 552 (for DOX) nm, respectively.