Doxorubicin

We added 960 µg of hydrogels (either 50 or 100 µm width) to 50 µL of a doxorubicin (LC Laboratories, Woburn, MA, USA) stock solution (1.5 mg/mL in water containing 0.1% v/v dimethyl sulfoxide (DMSO)) and left them for 24 h at room temperature. Unloaded doxorubicin was removed by careful aspiration of the supernatant and by washing once in 2 mL PBS.
To characterize the release of doxorubicin from the hydrogels, they were transferred to a 0.4 µm-pore filter insert for a 24-well plate and filled with 2 mL of PBS per well. At 4 h and then 1, 2, 3, 6 and 7 days, PBS was removed and stored (−80 °C) and replaced with fresh PBS. Then, 100 µL of the samples was analyzed by absorbance at 482 nm (Spark^® plate reader (Tecan Trading AG, Männedorf, Switzerland) in comparison to a standard curve as reported previously [25 (link)]. Four replicates were undertaken, and a mean value was used.
The doxorubicin-loaded hydrogels were also visualized via a fluorescence microscope set up for time-lapse imaging (Zeiss, Oberkochen, Germany; 10× lens). Images were acquired every hour for 48 h and analyzed using 4.8 AxioVision (Zeiss) with ImageJ (NIH) software (version 1.58j8) to determine the mean fluorescence intensity and create composite (brightfield + 488 nm) images.

HepG2 spheroids, alone or with LX2 or WI38 cells at a 1:1 ratio were formed for 3 days. After formation, the spheroids were treated with 10 μM doxorubicin (Sigma-Aldrich) for 1, 2, and 12 hr. For the image acquisition, each spheroids were fixed in 4% paraformaldehyde (Biosesang, Korea) for 30 min, and washed with Dulbecco’s phosphate-buffered saline (DPBS, Welgene) twice. The auto fluorescence of doxorubicin was detected using an inverted Zeiss laser scanning microscope (LSM) with a 100 W HBO mercury light source equipped with a 530 to 560 nm excitation and a 573 to 647 nm emission filter set. Spheroid images were captured in a 20 μm stack using 10× objectives. For measuring the absorbance of doxorubicin inside the spheroids, spheroids washed with DPBS twice in 1.5 ml tube after doxorubicin treatment for 1, 2, and 12 hr. After washing, each spheroid was transferred to 96-well and treated with 0.05% trypsin for 30 min at 37 °C incubator. After 30 min, spheroid in each well was pipetted smoothly until being dissociation to single cells. Using the 560 nm filter of spectrophotometer (Enspire, Perkin Elmer), absorbance of doxorubicin was measured.