4.4. P407 Hydrogel Degradation and Doxorubicin Release

Doxorubicin release was evaluated while using the ′membrane-less′ release method, as described in previous reports [27,28]. 1 mL of hydrogel mixed with 50 µg doxorubicin was loaded in a 15 mL falcon tube and then stored at 37 °C for 1 min. to induce gelation. Thereafter, 4 mL of 37 °C PBS was carefully layered on the gel layer. At the indicated time-points (2 h, 4 h, 6 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 120 h), 1 mL sample was withdrawn and the volume was replaced with 1 mL fresh PBS. After the final time-point, the samples were diluted and transferred to black 96-wells plates. Doxorubicin fluorescence (600 nm peak) was quantified with the Cytation 5 manager plate reader (BioTek® Instruments, Inc., Winooski, VT, USA). The doxorubicin standard curve was prepared under the same experimental conditions as the samples for release studies. In a separate analysis, it was evident that doxorubicin emission intensity in PBS and P407 solutions differs considerably (Appendix A: Table A1). Briefly, a known amount of doxorubicin was loaded in P407 hydrogel and PBS was layered on top. After one week, the hydrogel was fully degraded and this solution (end concentration 50 µg/mL doxorubicin) was diluted to create the calibration curve. The cumulative release was calculated according to Equation (1):

where E(%) is the cumulative release, V_E is the withdrawn volume (1 mL), V₀ is the begin volume (4 mL), C_i and C_n are the doxorubicin concentrations, i and n are the sampling times and m₀ is the total amount of doxorubicin loaded in the hydrogel (50 µg).

Alongside, hydrogel breakdown was measured. First 1 mL of hydrogel was loaded in 15 mL falcon tubes, and the difference in tube weight before and after loading is defined as 100% gel weight. Subsequently, 4 mL of PBS was carefully layered on top. At pre-determined time-points (corresponding to those from the release study), the entire volume was withdrawn and the change in falcon tube weight was measured to calculate the amount of broken down hydrogel.