To demonstrate drug release from the prodrug-conjugated hydrogels via controlled signal activation in live cell media, we supplied hydrogel patches containing the caged anticancer (GSH, Figure 'green triangle') (Figure ). GSH plays an important role in the cellular redox balance but also in cancer cell proliferation. Furthermore, GSH has been found at elevated levels in various human cancer tissues , which makes it a prime candidate for signaltriggered drug delivery. Gefitinib is an FDA approved reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, which is used for treating advanced non-small cell lung cancer, from which A549 cells are derived. We evaluated cell proliferation in the presence of prodrug-conjugated hydrogel patches during 72 hours wound healing assay via brightfield microscopy at incubator conditions. Proliferation results are shown in Figure , at t = 0 h and t = 72 h, where cellular wound closure can be used to qualitatively identify cell proliferation (wound closure) or inhibition (no wound closure) and furthermore quantify the change in cell density (from 0 to 72 h with time intervals of 6 h) during proliferation (Figure ). As expected, control experiments including a negative control (hydrogel only, no prodrug + 1% DMSO) and a positive control (hydrogel, no prodrug + 100 µM drug 6 in 1% DMSO) showed 97.8 ± 2.0% and 5.4 ± 12.3% wound closure after 72 h, respectively. The dextran-based hydrogel patch and 1% DMSO also show no cytotoxicity towards A549 cells. On the contrary, addition of 100 µM (46 µg/mL) of drug 6 leads to overall ~94% cell proliferation inhibition at the end of the observation period (Figure -positive control), which agrees with literature values. Next, we observed cell proliferation in the presence of hydrogel patches containing 5.0 mM conjugated prodrug 12 (0.04 eq. 12 vs alkyne units) and with or without signal-trigger drug activation (400 µM of 15). From the experimental results, we found a gradual wound closure increase within the first ~36h. Importantly, this was followed by a plateau with maxima at 25.2 ± 7.0% wound closure from 36 to 72 h (Figure We observed a lag time (~36 h, change in avg. cell density < 1%) in the wound healing assay during signal-triggered drug release, which is likely linked to the kinetics of the nucleophilic substitution reaction limited by diffusion to prodrug 12, and similarly to the diffusion of drug 6 from the hydrogel matrix. Indeed, such diffusion limitations related drug release lag time from a hydrogel matrix is often observed. 48 Additionally, we performed cell proliferation tests using only hydrogel, no prodrug and 400 µM of 15 (Supplementary Figure ), which showed no significant changes in cell proliferation compared to the positive control (Figure ).