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.
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.
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